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 Algae Refinery
Algae Refinery: Up- and Downstream Processes offers complete coverage of algae
refinery, including up- and downstream processes while proposing an integrated
algal refinery for the advancement of existing technologies and summarizing the
strategies and future perspectives of algal refinery. It provides a concise introduction
to the algal science, biology, technology, and application of algae. It explains
downstream and upstream steps of algal refinery for the production of algal biomass,
with several social benefits.

Features:

1. Provides various aspects of algal bioprocess including upstream and

downstream processes
2. Explains the major research streams of algae structures and their pathways
3. Covers algal-based CO2 capture technology
4. Explores the potential applications of algae for socioeconomical benefits
5. Deliberates algal bioremediation approach for clean and sustainable
development
Greener Technologies for Sustainable Industry and Environment
Series Editors
Pradeep Verma and Maulin P Shah

Greener technologies, such as microbial-based approaches and sustainable technolo-

gies with low net carbon output for energy generation, chemical production, bioreme-
diation, agriculture, and so forth, are preferable to less green alternatives. This series
attempts to provide space for scientists, chemical engineers, chemists, academicians,
industrialists, and environmentalists to bring out the best of the literature in their
area of expertise for developing future sustainable industry and environment.
Algae Refinery
Up- and Downstream Processes
Edited by Sanjeet Mehariya and Pradeep Verma

For more information about this series, please visit: www.routledge.com/Greener-Technologies-

For-Sustainable-Industry-And-Environment/book-series/GTSIE
 Algae Refinery
Up- and Downstream Processes

Edited by
Sanjeet Mehariya and Pradeep Verma
Designed cover image: © Shutterstock
First edition published 2024
by CRC Press
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and by CRC Press
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CRC Press is an imprint of Taylor & Francis Group, LLC
© 2024 selection and editorial matter, Sanjeet Mehariya and Pradeep
Verma; individual chapters, the contributors
Reasonable efforts have been made to publish reliable data and
information, but the author and publisher cannot assume responsibility
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ISBN: 978-1-032-52788-8 (hbk)
ISBN: 978-1-032-59718-8 (pbk)
ISBN: 978-1-003-45595-0 (ebk)
DOI: 10.1201/9781003455950
Typeset in Times
by Apex CoVantage, LLC
 Dedicated

To all contributors and future algal biotechnologists for their

contributions to sustainable development of algal biorefinery.
Contents
List of Figures��������������������������������������������������������������������������������������������������������� xiii
List of Tables������������������������������������������������������������������������������������������������������������ xv
Preface�������������������������������������������������������������������������������������������������������������������� xvii
Acknowledgments��������������������������������������������������������������������������������������������������� xxi
About the Editors�������������������������������������������������������������������������������������������������� xxiii
List of Contributors������������������������������������������������������������������������������������������������ xxv

Chapter 1 Introduction to Microalgae and Its Refinery���������������������������������������� 1

Udaypal, Rahul Kumar Goswami, Sanjeet Mehariya
and Pradeep Verma
1.1.Introduction������������������������������������������������������������������������������� 1
1.2. Classification and Overview of the Microalgae������������������������ 2
1.3. Upstream and Downstream Processing of Microalgae�������������� 4
1.3.1. Microalgae Cultivation�������������������������������������������������� 4
1.3.2. Downstream Processing������������������������������������������������ 6
1.4. Algae Biorefinery and Applications of Its Products������������������ 7
1.4.1. Bioenergy Products������������������������������������������������������� 7
1.4.2.Pharmaceuticals���������������������������������������������������������� 10
1.4.3.Cosmetics�������������������������������������������������������������������� 12
1.4.4.Chemicals�������������������������������������������������������������������� 12
1.4.5. Food products�������������������������������������������������������������� 13
1.4.6. Environmental Applications���������������������������������������� 15
1.5. Future Prospects and Conclusions������������������������������������������� 16
Acknowledgments������������������������������������������������������������������������������ 16
References������������������������������������������������������������������������������������������ 16

Chapter 2 Phycoremediation: A Sustainable Alternative for Removing

Emerging Contaminants from Wastewater����������������������������������������� 29
Preethi Selvaraj, Arathi Sreenikethanam, Subhisha Raj
and Amit K. Bajhaiya
2.1.Introduction����������������������������������������������������������������������������� 29
2.2. Emerging Contaminants (ECs)������������������������������������������������ 30
2.2.1. Organic Contaminants������������������������������������������������� 31
2.2.2. Inorganic Contaminants���������������������������������������������� 34
2.3. Methods for Removing Emerging Contaminants
from Wastewater���������������������������������������������������������������������� 35
2.3.1. Traditional Methods���������������������������������������������������� 35
2.3.2. Modern Methods��������������������������������������������������������� 41
2.4. Mechanism Used by Microalgae for Bioremediation������������� 42
2.4.1. Microalgal Biosorption of ECs����������������������������������� 42
vii
viii Contents

2.4.2. Bio-Uptake of ECs������������������������������������������������������ 42

2.4.3. Photodegradation and Volatilization��������������������������� 43
2.4.4. Biodegradation of ECs by Microalgae������������������������ 44
2.4.5.Bioaccumulation��������������������������������������������������������� 44
2.4.6. Co-culturing of Microalgae to Remove ECs��������������� 44
2.5.Conclusion������������������������������������������������������������������������������� 47
References������������������������������������������������������������������������������������������ 48

Chapter 3 Advances in Cultivation and Emerging Application of Chlorella

vulgaris: A Sustainable Biorefinery Approach����������������������������������� 54
Miriam L. Rosales-Aguado, Rosa M. Rodríguez-Jasso,
Samanta Machado-Cepeda, Gilver Rosero-Chasoy,
Regina Barboza-Rodríguez, Alejandra Cabello-Galindo
and Héctor A. Ruiz
3.1.Introduction����������������������������������������������������������������������������� 54
3.2. Chlorella vulgaris�������������������������������������������������������������������� 56
3.2.1. Growth Factors������������������������������������������������������������ 57
3.2.2. Environmental Factors������������������������������������������������ 60
3.2.3. Metabolic Pathways���������������������������������������������������� 62
3.2.4. Cultivation Systems����������������������������������������������������� 62
3.3. Culture Medium System���������������������������������������������������������� 63
3.3.1. Synthetic Mediums����������������������������������������������������� 64
3.3.2. Organic Mediums�������������������������������������������������������� 64
3.4. Biomass Harvesting����������������������������������������������������������������� 65
3.4.1.Centrifugation������������������������������������������������������������� 66
3.4.2.Flocculation����������������������������������������������������������������� 66
3.4.3.Flotation���������������������������������������������������������������������� 66
3.4.4.Filtration���������������������������������������������������������������������� 67
3.4.5.Sedimentation�������������������������������������������������������������� 67
3.5. Methods for Extraction������������������������������������������������������������ 67
3.6. Found in the Market with Different Applications�������������������� 69
3.6.1.Biofuels����������������������������������������������������������������������� 69
3.6.2. Human Nutrition��������������������������������������������������������� 69
3.6.3. Animal Feed���������������������������������������������������������������� 70
3.6.4. Cosmetology, Nutraceutical, and Pharmaceutical������� 70
3.7. Future Perspectives������������������������������������������������������������������ 71
3.8.Conclusion������������������������������������������������������������������������������� 71
Acknowledgments������������������������������������������������������������������������������ 71
References������������������������������������������������������������������������������������������ 72

Chapter 4 Algae Based Nutrient Recovery from Different Waste Streams�������� 79

Meenakshi Fartyal and Chitra Jain
4.1.Introduction����������������������������������������������������������������������������� 79
4.2. Algae and Their Role in Biotechnology���������������������������������� 80
4.3. Nutrients from Wastewater Streams���������������������������������������� 82
Contents ix

4.3.1. Municipal Wastewater������������������������������������������������� 84

4.3.2. Agricultural Wastewater���������������������������������������������� 85
4.3.3. Industrial Wastewater�������������������������������������������������� 86
4.4. Technologies to Recover Nutrients from Waste Streams�������� 86
4.4.1. Algae-Based Technologies������������������������������������������ 87
4.5. Mechanism of Nutrient Recovery�������������������������������������������� 89
4.5.1.Carbon������������������������������������������������������������������������� 89
4.5.2.Nitrogen���������������������������������������������������������������������� 90
4.5.3.Phosphorus������������������������������������������������������������������ 91
4.5.4. Other Nutrients������������������������������������������������������������ 91
4.6. Challenges and Limitations����������������������������������������������������� 92
4.7.Conclusions����������������������������������������������������������������������������� 93
Acknowledgments������������������������������������������������������������������������������ 93
References������������������������������������������������������������������������������������������ 93

Chapter 5 Potential Applications of Algae Biomass for the Development

of Natural Products��������������������������������������������������������������������������� 101
Getachew Tafere Abrha, Abdalah Makaranga, Bijaya Nag,
Gourav Kumar, Neeru Gupta, Asha Arumugam Nesamma
and Pannaga Pavan Jutur
5.1.Introduction��������������������������������������������������������������������������� 101
5.2. Algae-Based Energy Production������������������������������������������� 102
5.2.1.Biofuels��������������������������������������������������������������������� 103
5.2.2.Bioethanol����������������������������������������������������������������� 104
5.2.3.Biohydrogen�������������������������������������������������������������� 105
5.2.4.Biomethane��������������������������������������������������������������� 106
5.2.5.Biobutanol����������������������������������������������������������������� 107
5.3. Biopotential of Algae-Based Products����������������������������������� 108
5.3.1. Polyunsaturated Fatty Acids (PUFAs)����������������������� 108
5.3.2.Sterols������������������������������������������������������������������������ 109
5.3.3.Carotenoids��������������������������������������������������������������� 109
5.3.4.Polysaccharides��������������������������������������������������������� 109
5.3.5.Vitamins�������������������������������������������������������������������� 110
5.3.6. Microalgal Proteins��������������������������������������������������� 110
5.3.7.Phycobiliproteins������������������������������������������������������ 110
5.3.8. Livestock and Agriculture����������������������������������������� 111
5.4. Algae-Based Companies������������������������������������������������������� 112
5.5. Conclusions, Challenges, and Future Perspectives��������������� 113
Acknowledgments���������������������������������������������������������������������������� 116
References���������������������������������������������������������������������������������������� 116

Chapter 6 Algal Metal Remediation for Contaminated Source������������������������ 126

Alka Rani and Khem Chand Saini
6.1.Introduction��������������������������������������������������������������������������� 126
6.2. Sources of Heavy Metals (HMs)������������������������������������������� 127
x Contents

6.3. Impact of HMs����������������������������������������������������������������������� 127

6.3.1. Effects on Soil����������������������������������������������������������� 128
6.3.2. Effects on Water�������������������������������������������������������� 129
6.3.3. Effects on Air������������������������������������������������������������ 129
6.3.4. Effects on Aquatic Ecosystem����������������������������������� 129
6.4. Phycoremediation: An Algal Mechanism to Eradicate
Pollution�������������������������������������������������������������������������������� 129
6.4.1. Extracellular Uptake (Biosorption)��������������������������� 130
6.4.2. Intracellular Uptake (Bioaccumulation and
Compartmentalization)���������������������������������������������� 134
6.5. Strategies to Improve the Bioremediation Ability of Algae����� 134
6.5.1. Algal Metal Transportation��������������������������������������� 136
6.5.2. Metal Chelation��������������������������������������������������������� 137
6.5.3. Metal Biotransformation������������������������������������������� 138
6.5.4. Oxidative Stress Response Regulation���������������������� 138
6.5.5. Metal Stress Response Regulation���������������������������� 139
6.5.6. Bioengineering of Algal Cell Surface����������������������� 139
6.6. Conclusion and Future Perspective��������������������������������������� 140
References���������������������������������������������������������������������������������������� 140

Chapter 7 Algal–Bacterial Interactions in Environment:

Emerging Applications��������������������������������������������������������������������� 148
Imran Pancha
7.1.Introduction��������������������������������������������������������������������������� 148
7.2. Microalgal Bacteria Interactions in Natural Environments��� 150
7.3. Biotechnological Applications of Microalgal–Bacterial
Interactions���������������������������������������������������������������������������� 153
7.4. Conclusion and Future Prospects������������������������������������������ 156
Acknowledgements�������������������������������������������������������������������������� 156
References���������������������������������������������������������������������������������������� 157

Chapter 8 Sustainable Bio-Applications of Diatom Silica as

Nanoarchitectonic Material�������������������������������������������������������������� 161
Sahil Kapoor, Meenakshi Singh, Sanchita Paul, Surojit Kar,
Trisha Bagchi, Murthy Chavali and K. Chandrasekhar
8.1.Introduction��������������������������������������������������������������������������� 161
8.2. Diatomaceous Nanostructures – A Living Source of
Biogenic Silica����������������������������������������������������������������������� 164
8.2.1. Biophysical Properties���������������������������������������������� 164
8.2.2. Mechanical Properties����������������������������������������������� 165
8.2.3. Chemical Properties�������������������������������������������������� 165
8.2.4. Optical Properties������������������������������������������������������ 166
8.2.5. Electronic Properties������������������������������������������������� 166
8.2.6. Metallurgical Properties�������������������������������������������� 167
Contents xi

8.3. Scientometric Analysis���������������������������������������������������������� 168

8.4. Nanofabrication Techniques to Prepare Hierarchical
Biosilica Matrix��������������������������������������������������������������������� 169
8.4.1. Atomic Force Microscopy (AFM)���������������������������� 169
8.4.2. Transmission Electron Microscopy (TEM)��������������� 169
8.4.3. X-Ray Photoelectron Spectroscopy (XPS)��������������� 170
8.4.4. Surface-Enhanced Raman Scattering (SERS)����������� 171
8.4.5. Fourier-Transform Infrared Spectroscopy (FTIR)���� 171
8.4.6. X-Ray Powder Diffraction (XRD)���������������������������� 172
8.5. Application Based on Diatoms Silica Nanomaterials����������� 172
8.5.1.Biotemplates�������������������������������������������������������������� 172
8.5.2.Bioprinting���������������������������������������������������������������� 173
8.5.3.Biosensors����������������������������������������������������������������� 174
8.5.4.Biofiltration��������������������������������������������������������������� 174
8.5.5.Biocomposites����������������������������������������������������������� 176
8.5.6. Biomimetic Analogues���������������������������������������������� 176
8.5.7. Biomanufacturing Technology���������������������������������� 177
8.6. Challenges Encountered in Diatom-Inspired
Nanostructure Technologies�������������������������������������������������� 177
8.6.1. Photonic Nanotechnology����������������������������������������� 177
8.6.2. Bioreactor Nanotechnology�������������������������������������� 178
8.7.Conclusion����������������������������������������������������������������������������� 179
Authorship Contribution������������������������������������������������������������������� 179
References���������������������������������������������������������������������������������������� 180

Chapter 9 Algal Biofuel: A Promising Source of Green Energy���������������������� 187

Prachi Agrawal, Kushal Kant Pant, Madan Sonkar
and Vikas Chandra
9.1.Introduction��������������������������������������������������������������������������� 187
9.2.Algae�������������������������������������������������������������������������������������� 188
9.3. Cultivation of Microalgae������������������������������������������������������ 192
9.3.1. Closed System����������������������������������������������������������� 193
9.3.2. Open System������������������������������������������������������������� 193
9.3.3. Hybrid System����������������������������������������������������������� 194
9.4. Harvesting of Microalgae������������������������������������������������������ 194
9.5. Algal Biofuels������������������������������������������������������������������������ 194
9.5.1. Biodiesel Production������������������������������������������������� 195
9.5.2. Bioethanol Production����������������������������������������������� 197
9.5.3. Biogas Production����������������������������������������������������� 198
9.5.4. Biohydrogen Production������������������������������������������� 198
9.5.5. Bio-Oil and Syngas Production��������������������������������� 198
9.6. Current Status and Bottlenecks��������������������������������������������� 199
9.7.Conclusion����������������������������������������������������������������������������� 200
Competing Interest��������������������������������������������������������������������������� 201
References���������������������������������������������������������������������������������������� 201
xii Contents

Chapter 10 Life Cycle Assessment (LCA), Techno-Economic Analysis

(TEA) and Environmental Impact Assessment (EIA)
of Algal Biorefinery�������������������������������������������������������������������������� 209
Bikash Kumar, Tonmoy Ghosh, Sukhvinder Singh Purewal
and Kiran Bala
10.1.Introduction��������������������������������������������������������������������������� 209
10.2. General Overview of Life Cycle Assessment������������������������ 211
10.3. Tools Used for the LCA and Impact Assessment Analysis����� 213
10.3.1.SimaPro��������������������������������������������������������������������� 213
10.3.2.openLCA������������������������������������������������������������������� 213
10.3.3. One Click LCA��������������������������������������������������������� 213
10.3.4.GaBi�������������������������������������������������������������������������� 214
10.3.5. BEES (Building for Environmental
and Economic Sustainability)����������������������������������� 214
10.3.6.esg.tech���������������������������������������������������������������������� 214
10.3.7 Ecoinvent Database��������������������������������������������������� 214
10.4. Methods, Framework, and LCA and LCIA of the
Algal-Biorefinery������������������������������������������������������������������� 215
10.4.1. Component and Parameters for LCA of Algal
Biorefinery���������������������������������������������������������������� 215
10.5. Comprehensive Reviews of LCA and LCIA for Different
Algal Biorefineries Processes������������������������������������������������ 221
10.6. LCA of the Microalgae-Based Biorefinery Supply
Network and the Need for Integrated Biorefineries�������������� 224
10.7. Role of LCA and LCIA in Policy Decisions Based on
Algal Biorefineries���������������������������������������������������������������� 225
10.8.Conclusions��������������������������������������������������������������������������� 226
Competing Interest��������������������������������������������������������������������������� 226
Funding & Acknowledgment����������������������������������������������������������� 227
References���������������������������������������������������������������������������������������� 227

Index���������������������������������������������������������������������������������������������������������������������� 237
Figures
Figure 1.1 G
 eneral representation of types of (a) microalgae cultivation
systems and (b) types of microalgae cultivation modes.�������������������� 4
Figure 1.2 U
 pstream cultivation and downstream processing
of microalgae biomass.����������������������������������������������������������������������� 6
Figure 1.3 M
 icroalgae biomass-based biorefineries and different high
value-added products.����������������������������������������������������������������������� 12
Figure 2.1 M
 ethods for removing emerging contaminants
from wastewater.������������������������������������������������������������������������������� 35
Figure 2.2 I on exchange method to remove emerging contaminants
from wastewater.������������������������������������������������������������������������������� 36
Figure 2.3 E
 lectrochemical methods to remove emerging contaminants
from wastewater.������������������������������������������������������������������������������� 37
Figure 2.4 Fungi-assisted microalgae bio-flocculation method.������������������������ 45
Figure 3.1 General diagram process of Chlorella vulgaris production.������������� 56
Figure 3.2 A typical photograph taken of Chlorella using a microscope.��������� 57
Figure 3.3 Scientific classification of Chlorella vulgaris.��������������������������������� 60
Figure 3.4 Principal growth factors that affect Chlorella vulgaris.������������������� 60
Figure 4.1 Nutrient recovery technology.���������������������������������������������������������� 83
Figure 4.2 Microalgae–bacteria consortium.����������������������������������������������������� 87
Figure 5.1 Schematic presentation of biofuel production from microalgae.��� 103
Figure 6.1 B
 ioaccumulation and detoxification of heavy metals in algae
using transporters. Abbreviations: MTP, metal tolerance
protein; ZIP, Zrt-Irt-like proteins; NRAMP, natural resistance-
associated macrophage proteins; MATE, multidrug and toxic
compound extrusion; ABC transporters, ATP-binding cassette
transporters; CTR, Cu transporter.�������������������������������������������������� 136
Figure 7.1 Microalgal–bacterial interactions and its potential applications.�������154
Figure 8.1 T
 he nanoarchitectonics methodology for fabricating functional
materials from nanoscale components.������������������������������������������� 162
Figure 8.2 The internal structural arrangement of a diatom.��������������������������� 165
Figure 8.3 T
 he diatom biosilica reflects the light, and its porosity enables
light–frustule interaction to diffract and scatter, which is
captured by a spectrophotometer.��������������������������������������������������� 167

xiii
xiv Figures

Figure 8.4 Scientometric analysis on diatom-based nanoarchitectonics.��������� 168

Figure 8.5 D
 iatom fabrication with metal ions by EDTA treatment and
the study of growth analysis using TEM, XRD, EDX, UV/VIS
spectrophotometry, and FTIR.�������������������������������������������������������� 179
Figure 9.1 Various generations of biofuels.����������������������������������������������������� 188
Figure 9.2 Different types of biofuels produced from algal biomasses.���������� 189
Figure 9.3 Advantages of algal biomass for the production of biofuels.��������� 191
Figure 9.4 D
 ifferent processes used for the conversion of algal biomass
into useful products/energy.������������������������������������������������������������ 192
Figure 9.5 Different algal cultivation techniques for biofuel production.������� 193
Figure 10.1 Potential application of algae.���������������������������������������������������������211
Figure 10.2 Life cycle assessment framework.�������������������������������������������������� 212
Figure 10.3 L
 ife cycle data inventory for algal biorefinery (production of
biodiesel, succinic acid, and algal protein).������������������������������������ 216
Figure 10.4 F
 ormulas and explanation for different environmental impact
assessment parameters.������������������������������������������������������������������� 217
Figure 10.5 E
 nvironmental, health impact of 19 impact categories from five
cultivation steps of dried seaweed (S. latissima) (as shared by
Creative Common license (Nilsson et al. 2022)).��������������������������� 219
Tables
Table 1.1 B rief Classifi cations and Overview of Commercially Important
Microalgae and Their Reported Products.�������������������������������������������� 3
Table 1.2 Protein, Lipid, and Carbohydrate Composition of Commercially
Important Microalgae.������������������������������������������������������������������������ 14
Table 2.1 Types of Emerging Contaminants and the Phycoremediation
Efficiency.������������������������������������������������������������������������������������������� 31
Table 3.1 Chlorella spp. within Different Approaches.�������������������������������������� 58
Table 3.2 Different Extraction Methods Employed on Chlorella vulgaris.������� 68
Table 4.1 Total Nitrogen (TN) and Total Phosphorus (TP) Content of
Different Waste Streams.�������������������������������������������������������������������� 85
Table 5.1 Bioactive Compounds with Potential Applications from
Different Algae.���������������������������������������������������������������������������������111
Table 5.2 Various Companies Produce Algae-Based Biofuels
and Value-Added Products.���������������������������������������������������������������114
Table 6.1 Sources of HMs, Their Acceptable Level, and Their Impact
on Human Health.����������������������������������������������������������������������������� 128
Table 6.2 Algal Species Used for the Removal of the Heavy Metals.������������� 131
Table 6.3 List of Transporter Families and Genetic Engineering Targets
Foreseen to Improve the Capacity of Microalgae to
Bioremediate HMs.��������������������������������������������������������������������������� 135
Table 8.1 Bio-Functional Properties of Diatom-Based Silica Applications.���� 163
Table 8.2 Summary of Diatom Frustules Acting as Silica-Based
Biosensors for Nanomaterials.���������������������������������������������������������� 175
Table 9.1 Differentiation of Algal Groups Based on Different
Characteristics (Gamal Saad etal. 2019)������������������������������������������ 189
Table 10.1 Life Cycle Impacts Assessment Results for 19 Impact Categories
for 1 kg Dry and 1 kg Wet Seaweed (Nilsson et al., 2022)�������������� 218
Table 10.2 Comprehensive Summary of Sensitivity Analysis, Functional
Unit, Allocation Technique, Global Warming Potential, and
Impact Category for LCA System Boundary.����������������������������������� 222

xv
Preface
Algae are a large and diverse group of single-celled/multi-celled autotrophic
organisms existing in a variety of environments. In recent years, due to increasing
industrialization and urbanization, rapid climate change is occurring. The uneven
rains, seismic movements, cyclones, etc., are impacting agriculture production,
thus impacting food and feed production. Also, water pollution by rapid growth has
become a critical problem along with a lack of resources to meet global energy and
chemical needs. The algal-based biorefinery thus can act as a potential solution to
this existing problem of lack of natural resources and pollution and climate change.
Thus, intensive and integrated research on algal biorefinery is highly required in
the upstream and downstream processes.
With contributions from world experts, this book focuses on algae refinery from
the upstream and downstream processes. In addition, it proposes an integrated algal
refinery for the advancement of existing technologies. Also, it summarizes the strat-
egies and future perspectives of the microalgal refinery to integrate with circular
bioeconomy concepts.
Chapter 1 discusses the introduction to photosynthetic algae, classification, and
general overview of microalgae. Moreover, a brief overview of upstream and down-
stream processes such as cultivation, harvesting, drying, and extraction has been
deliberated. Moreover, the applications of microalgae and its biomass in bioenergy,
pharmaceuticals, and food production as well as environmental benefits have been
discussed. Furthermore, this book chapter focuses on the utilization of microalgae as
an effective and economically feasible feedstock for biorefineries.
Chapter 2 discusses the issue related to surface water pollution associated with
emerging contaminants and their algal-based removal. The concentration of these
contaminants in different water bodies ranges from micrograms to milligrams per
liter of wastewater. These pollutants can be either organic or inorganic including
pharmaceutical compounds, industrial chemicals, endocrine disruptors, and heavy
metals. Removal of these contaminants is one of the biggest challenges of the era.
Many traditional and modern techniques are employed to eliminate these contam-
inants from wastewater, but each have their limitations. The higher-energy input
requirements of traditional techniques including ion exchange, electrochemical treat-
ment, osmosis, evaporation, and precipitation make them more expensive for appli-
cation on a large scale. However, modern techniques based on biological processes
(ex. Bioremediation) are bringing new hopes of a sustainable, simple, and green way
of removing these pollutants from wastewater. Referring to several studies, one of
the most effective strategies to eliminate emerging pollutants from wastewater is to
employ microalgae for bioremediation. Microalgae are eukaryotic, photosynthetic,
ubiquitous microscopic organisms that can thrive in almost all kinds of water bod-
ies. They are reported to be bioremediate heavy metals and emerging contaminants
by the mechanism of bio-assimilation and biosorption. Algae-based treatments have
become an efficient, economical, and low-energy input approach compared to chem-
ical treatments. The application of microalgae for the bioremediation of emerging

xvii
xviii Preface

organic and inorganic contaminants from wastewater will be discussed in this chap-
ter. Further, the impact of these emerging pollutants on the environment and the
phytoremediation capabilities of algae will also be discussed.
Chapter 3 describes the advances in cultivation and emerging application of
microalgae Chlorella vulgaris. Chlorella vulgaris is a green microalga of great inter-
est for the components found in its biomass; some of these components are proteins,
carbohydrates, and lipids; also, the pigments it contains are considered high-value-
added products. In recent years, the study and application of this microalga have
increased for the energy, pharmaceutical, cosmetic, and food industry and applica-
tions. Therefore, this chapter aims to emphasize the emerging technologies devel-
oped under the biorefinery concept using various alternatives of culture media, like
synthetic medium supplemented with agro-industrial residues, whose organic matter
is an essential source of nutrition for biomass growth and is also necessary for the
accumulation of the high-added-value components. Different strategies for recover-
ing microalgal biomass are discussed such as centrifugation, flotation, flocculation,
filtering, and sedimentation. The methods of extracting high-value-added com-
pounds, such as the subjection of biomass to freezing cycles, are also mentioned. It
also reviews the systems used for cultivation, such as open ponds, raceways, and pho-
tobioreactors, for the development of third-generation biorefineries with an impact
on the circular bioeconomy and process sustainability.
Chapter 4 explores how algae are important for nutrient recovery from different
waste streams. A national trend to lower the permissible nutrient levels in waste-
water treatment plant (WWTP) outflow is provoking facility owners to think about
new treatment approaches like algae. The assessed sensitivity of nearby water bodies
presently determines the amount of fertilizer loading permitted downstream. Algae
have the potential to play a significant part in helping facilities manage their side-
stream treatment to meet strict downstream requirements, reduce their total effluent
nutrients, and minimize overall plant operating costs. Algae, often thought to be
an annoyance for WWTPs, may now be essential to resource recovery and nutrient
management. Algae may be used to remediate wastewater and have several benefits.
Algae are easily accessible, highly flexible, and able to significantly absorb nutrients.
The technique is particularly suitable for areas with high levels of sunlight and high
temperatures because photosynthesis is essential for the development of microalgae
and requires minimal upkeep. Algae offer a different route, and when used, it pro-
duces high-quality treated water. The requirement for big footprints and the high
cost/energy associated with algae harvesting are major obstacles to the widespread
use of algae in wastewater treatment. University–industry research collaboration is
coming to notice, though, for its pioneering method of cultivating and collecting the
algae in a wastewater application with a less environmental impact.
Chapter 5 highlights the potential of the microalgae cell factories for developing
various valuable products which find wide application in different fields. This chap-
ter explains the potential of algae biomass application as a biorenewable feedstock
to produce biofuels and their subsequent utilization as a potential source of pharma-
ceuticals and high-value products; it could be an option for a sustainable solution for
making a better global environment and economy.
Preface xix

Chapter 6 discusses how algae are a group of microorganisms that are able to be
used for metal remediation from contaminated sources. The fast-growing industri-
alization and anthropogenic activities, including fossil fuel burning, unmanaged use
of agrochemicals, and the release of sewage sludge, trigger soils and water bodies
to be harshly contaminated with persistent and nonbiodegradable heavy metals that
threaten all life forms. These reactive metals accumulate in the food web, instigating
severe health concerns. Diverse modern techniques such as ion exchange, chemical
extraction, and electrolytic technologies have been adapted for the remediation of
heavy metals, but these techniques are neither economical nor sustainable; instead,
they need continuous monitoring and stringent control. Therefore, this chapter sum-
marizes the information on algal strains used for eliminating metal contamination and
the mode of action they followed for heavy metals remediation. Algal strains, namely
Anabaena spp., Chlorella spp., Cladophora spp., Spirulina spp., Scenedesmus spp.,
Oscillatoria spp., Phaeodactylum tricornutum, etc., have exhibited the capability
to eradicate heavy metals, acting as hyper-adsorbents and hyper-accumulators with
high selectivity for various metals. Phycoremediation alone is not economical; there-
fore, integrated remediation systems must be developed, making this approach more
sustainable and economical. Furthermore, this review explores current progressions
in the phycoremediation of heavy metals. Also discussed is the genetic engineer-
ing approach applied to create transgenic species leading to the over-expression of
metallothioneins and phytochelatins that form complexes with heavy metals and are
stored within the vacuoles utilizing bioaccumulation to remove hazardous metals.
Following the discussion, this chapter concluded that phycoremediation is a more
bioeconomic, sustainable, and clean technology.
Chapter 7 describes how algal–bacterial interactions in the environment have var-
ious applications. In the natural environment, microalgae are not alone, as most of
the time they are found in consortia with various microorganisms such as bacteria
and fungi. Therefore, their interaction with other organisms such as bacteria plays
a very important role in various processes such as biogeochemical cycles, nutrient
recycling, wastewater treatment, and sustainable agriculture. Understanding such
interaction helps us to develop processes for various biotechnological applications.
In this book chapter, we will discuss our understanding of microalgae–bacteria inter-
action in the natural environment and its probable applications such as the production
of sustainable biomass and wastewater remediation.
Chapter 8 explains the application of diatoms, which are the living factories of
biogenic porous silica. The frustules have an inimitable architecture of intricate
nanopatterns that are biocompatible, thermally stable, and corrosion- and pathogen
resistant. Diatom silica microparticles have inspired multiple nanofabrication tech-
niques, which are sustainable, economical, environmentally benign, and efficient
in various nano-designed biosystems. Diatom-based nanoarchitectonics is a novel
nano-scale approach in material science, which works under complex physiologi-
cal conditions. This review focuses on such potential bioapplications and their tools
to prepare hierarchical silica nanostructures to tailor multifunctional properties
for improved microfabrication and functional design. A brief account of sciento-
metric analysis on diatom silica as advanced nanoarchitectonics material to create
xx Preface

molecular machines and micro devices is described. Moreover, the challenges faced
in diatom-based nanostructure technologies are also discussed.
Chapter 9 explores the biofuel production by algal biomass; the production of
algal biofuels is one such renewable alternative to overcome this global crisis. Algae
being flourishing organisms are proficient of growth and expansion under variable
conditions like controlled environment in closed photobioreactors as well as in open
pond ecosystems. Also, alteration in their optimal growth conditions and genetic
alterations result in varied secretion and aggregation of biofuels. Bio-transforming
the algal biomass in distinct forms of biofuels can be a key step to solving the prob-
lem of growing energy demand. Although algal biofuel serves as a substitute to
conventional fossil fuel, the production technology needs to conquer commercial
barricades and various challenges that obstruct the production of such green energy
solutions. A superior understanding of basic procedures involved in biofuel produc-
tion is needed. In this chapter, a thorough discussion is made on different biofuels
produced by algae with major challenges that obstruct productivity. In addition, it
also enlightens the advancements made to meliorate the productivity of biofuels.
Chapter 10 describes a market survey and trend to increase the global demand
and consumption of algal biomass for various applications. Therefore, this chapter
emphasizes the developing fields of algal science with a specific focus on algal diver-
sity, digestibility, bioavailability, active bioactive components, and applications that
are required for a better assessment of the health benefits of these algae or algal
by-products. Furthermore, vast opportunities exist for phycologists, other research-
ers, and people interested in developing new products and their industrial applica-
tions in this emerging field.
Overall, this book covers various aspects of algal biorefinery, including the
upstream and downstream processes.

Sanjeet Mehariya, PhD

Pradeep Verma, PhD
Acknowledgments
First and foremost, the editors would like to express their sincere gratitude to all the
distinguished authors for their thoughtful contribution to making this project suc-
cessful. We appreciate their patience and diligence in revising the first draft of the
chapters after assimilating the suggestions and comments. We would like to thank
and acknowledge the solicitous contributions of all the reviewers who spent their
valuable time in providing constructive comments to improve the quality of the chap-
ters. Finally, we would like to acknowledge the support of our mentors, family mem-
bers, friends, and colleagues for their love and encouragement; this work is dedicated
to their smiles.

xxi
About the Editors
Sanjeet Mehariya (PhD) is Researcher at the Algal
Technology Program, Center for Sustainable Development,
College of Arts and Sciences, Qatar University, Doha, Qatar.
Also, Dr Mehariya worked as Postdoctoral Researcher at the
Department of Chemistry, Umeå University, Umeå, Sweden.
Dr. Mehariya earned a PhD in Engineering at the University
of Campania “Luigi Vanvitelli”, Italy. He has collaborated
with national and international stakeholders, including pol-
icymakers, industries, and prominent RTD institutes in the
field of biobased economy. He has worked at CSIR-Institute
of Genomics and Integrative Biology, Delhi, India; Konkuk University, Seoul,
South Korea; ENEA-Italian National Agency for New Technologies, Energy and
Sustainable Economic Development, Rome, Italy; Hong Kong Baptist University,
Hong Kong; University of Campania “Luigi Vanvitelli”, Italy; and Sapienza—
University of Rome, Italy. He has published more than 80 research and review arti-
cles and has edited four books. He is a life member of various societies such as
Scandinavian Plant Physiology Society (SPPS), Sweden; Biotech Research Society
(BRSI), India; the Association of microbiologist of India (AMI); Life Indian Science
Association (ISA); and International Forum for BOTANISTS Under the auspices of
ISLS, India. Dr. Mehariya serves as a peer review member assessor for the Horizon
H2020-Marie Sklodowska-Curie Individual Fellowships-Europe.

Pradeep Verma (PhD) is currently working at the Department

of Microbiology, Central University of Rajasthan (CURAJ),
Ajmer, India. Prof. Verma completed his PhD from Sardar
Patel University, Gujarat, India, in 2002. He was selected
as UNESCO fellow (2002) and joined Czech Academy of
Sciences, Prague, Czech Republic. He later moved to Charles
University, Prague, to work as Post-Doctoral Fellow. He joined
as Visiting Scientist (2004) at UFZ Centre for Environmental
Research, Halle, Germany. He was awarded a DFG fellowship
(2004) to work as Post-Doctoral Fellow at Gottingen University, Germany. He moved
to India in 2007 where he joined Reliance Life Sciences, Mumbai, and worked
extensively on biobutanol production which attributed a few patents to his name.
Later, he was awarded with JSPS Post-Doctoral Fellowship Programme and joined
Laboratory of Biomass Conversion, Research Institute of Sustainable Humanosphere
Kyoto University, Japan. He is also a recipient of various prestigious awards such as
Ron-Cockcroft award by Swedish society and UNESCO Fellow ASCR Prague. Prof.
Verma is Fellow of many prestigious societies in India, for example, BRSI, MSI,
and AMI. Prof. Verma is Group Leader of Bioprocess and Bioenergy Laboratory,
Department of Microbiology, Central University of Rajasthan, Bandarsindri,
Kishangarh, Ajmer, Rajasthan, India, and his area of expertise involves Bioprocess
Development and Lignocellulosic and Algal Biomass-Based Biorefinery.
xxiii
Contributors
Getachew Tafere Abrha Vikas Chandra
Omics of Algae Group, International Department of Biotechnology, Guru
Centre for Genetic Engineering and Ghasidas Central University,
Biotechnology, Aruna Asaf Ali Marg Bilaspur
New Delhi, India Chhattisgarh, India

Prachi Agrawal K. Chandrasekhar

Department of Biotechnology, Guru
Ghasidas Central University, Bilaspur
Chhattisgarh, India
Trisha Bagchi
Department of Botany, West Bengal State
University, Berunanpukuria, Malikapur,
Barasat, 24 Parganas (North)
West Bengal, India
Amit K. Bajhaiya
Algal Biotechnology Lab, Department
of Microbiology, School of Life
Sciences, Central University of Tamil
Nadu, Thiruvarur
Tamil Nadu, India
Department of Biotechnology, Vignan’s
Foundation for Science, Technology
and Research, Vadlamudi, Guntur
Andhra Pradesh, India
Murthy Chavali
MIT World Peace University, Kothrud,
Pune
Maharashtra, India
Meenakshi Fartyal
Department of Biotechnology, Kanoria
PG Mahila Mahavidyalaya,
University of Rajasthan, Jaipur
Rajasthan, India

Kiran Bala Tonmoy Ghosh

Department of Biosciences and Biomedical Institute for Water and Wastewater
Engineering, Indian Institute of Technology, Durban University of
Technology Indore, Simrol, Indore Technology
Madhya Pradesh, India Durban, South Africa

Regina Barboza-Rodríguez Rahul Kumar Goswami

Biorefinery Group, Food Research
Department, School of Chemistry,
Autonomous University of Coahuila,
Saltillo
Coahuila, Mexico
Alejandra Cabello-Galindo
Biorefinery Group, Food Research
Department, School of Chemistry,
Autonomous University of Coahuila,
Saltillo
Coahuila, Mexico
Bioprocess and Bioenergy Laboratory
(BPBEL), Department of
Microbiology, Central University of
Rajasthan, Bandarsindri, Kishangarh,
Ajmer
Rajasthan, India
Neeru Gupta
Omics of Algae Group, International
Centre for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg
New Delhi, India

xxv
xxvi
Chitra Jain
Biomitra Life Sciences Private Limited,
Jaipur
Rajasthan, India
Pannaga Pavan Jutur
Omics of Algae Group, International
Centre for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg
New Delhi, India
Sahil Kapoor
Department of Botany, Goswami
Ganesh Dutta S.D. College
Chandigarh, India
Surojit Kar
Department of Botany, West Bengal
State University, Berunanpukuria,
Malikapur, Barasat, 24 Parganas
(North)
West Bengal, India
Bikash Kumar
Department of Biosciences and
Biomedical Engineering, Indian
Institute of Technology Indore,
Simrol, Indore
Madhya Pradesh, India
Gourav Kumar
Omics of Algae Group, International
Centre for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg
New Delhi, India
Samanta Machado-Cepeda
Biorefinery Group, Food Research
Department, School of Chemistry,
Autonomous University of Coahuila,
Saltillo
Coahuila, Mexico
Abdalah Makaranga
Omics of Algae Group, International
Centre for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg
New Delhi, India
Contributors
Sanjeet Mehariya
Algal Technology Program, Center for
Sustainable Development, College of
Arts and Sciences, Qatar University
Doha, Qatar
Bijaya Nag
Omics of Algae Group, International
Centre for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg
New Delhi, India
Asha Arumugam Nesamma
Omics of Algae Group, International
Centre for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg
New Delhi, India
Imran Pancha
Department of Industrial Biotechnology,
Gujarat Biotechnology University,
Gandhinagar
Gujarat, India
Kushal Kant Pant
Department of Biotechnology, Guru
Ghasidas Central University,
Bilaspur
Chhattisgarh, India
Sanchita Paul
Department of Botany, West Bengal State
University, Berunanpukuria, Malikapur,
Barasat, 24 Parganas (North)
West Bengal, India
Sukhvinder Singh Purewal
University Centre for Research &
Development (UCRD), Chandigarh
University, Mohali
Punjab, India
Subhisha Raj
Algal Biotechnology Lab, Department
of Microbiology, School of Life
Sciences, Central University of Tamil
Nadu, Thiruvarur
Tamil Nadu, India
Contributors
Alka Rani
Department of Botany, School of Basic
and Applied Sciences, Central
University of Punjab, Bathinda
Punjab, India
Rosa M. Rodríguez-Jasso
Biorefinery Group, Food Research
Department, School of Chemistry,
Autonomous University of Coahuila,
Saltillo
Coahuila, Mexico
Miriam L. Rosales-Aguado
Biorefinery Group, Food Research
Department, School of Chemistry,
Autonomous University of Coahuila,
Saltillo
Coahuila, Mexico
Gilver Rosero-Chasoy
Biorefinery Group, Food Research
Department, School of Chemistry,
Autonomous University of Coahuila,
Saltillo
Coahuila, Mexico
Héctor A. Ruiz
Biorefinery Group, Food Research
Department, School of Chemistry,
Autonomous University of Coahuila,
Saltillo
Coahuila, Mexico
Khem Chand Saini
School of Basic & Applied Sciences,
Nirwan University, Jaipur
Rajasthan, India
xxvii
Preethi Selvaraj
Algal Biotechnology Lab, Department
of Microbiology, School of Life
Sciences, Central University of Tamil
Nadu, Thiruvarur
Tamil Nadu, India
Meenakshi Singh
Department of Ecology & Biodiversity,
Sustaina Greens LLP, Vadodara
Gujarat, India
Madan Sonkar
Department of Biotechnology, Guru
Ghasidas Central University, Bilaspur
Chhattisgarh, India
Arathi Sreenikethanam
Algal Biotechnology Lab, Department
of Microbiology, School of Life
Sciences, Central University of Tamil
Nadu, Thiruvarur
Tamil Nadu, India
Udaypal
Bioprocess and Bioenergy Laboratory
(BPBEL), Department of
Microbiology, Central University of
Rajasthan, Bandarsindri, Kishangarh,
Ajmer
Rajasthan, India
Pradeep Verma
Bioprocess and Bioenergy Laboratory
(BPBEL), Department of Microbiology,
Central University of Rajasthan,
Bandarsindri, Kishangarh, Ajmer
Rajasthan, India
 1 Introduction to
Microalgae and
Its Refinery
Udaypal, Rahul Kumar Goswami,
Sanjeet Mehariya and Pradeep Verma

1.1 INTRODUCTION
Algae are a wide group of microorganisms that vary in size from microscopic blue–
green algae to massive, complex seaweeds that may be a few meters in length (Singh
and Saxena 2015). Compared to terrestrial plants, algae possess a wide variety of
photosynthetic processes, particularly in the form of pigments and structures that
capture light and fix carbon dioxide (CO2) (Larkum et al. 2003). They are photoau-
totrophic, having a simple thallus and no tissue differentiation. They can be found in
a variety of aquatic environments, including harsh environments and fresh as well
as saline water. There is a lot of variation in their reproduction and perennation
methods due to their diversity in structure and habitat (Sahoo and Baweja 2015). The
term “algae” includes both macroalgae (large size) and the extremely broad class of
microorganisms known as microalgae (microscopic size). According to estimates,
there are millions of different kinds of algae, the majority of which are microalgae
(Evangelista et al. 2008).
Microalgae are unicellular photosynthetic organisms that often are found in
freshwater and marine aquatic habitats. The size range of microalgae is 0.2 to 2
µm (picoplankton) up to filamentous forms that are 100 µm or larger (Ghosh et al.
2021). They can be cultivated in both open and closed systems in diverse cultivation
modes, that is, photoautotrophic, heterotrophic, and mixotrophic. Depending on the
species, microalgae can produce a variety of co-products (Gara and Stark 2012).
Due to the co-production of additional molecules like pigments, proteins, polyun-
saturated fatty acids (PUFAs), and antioxidants, microalgae are gaining significant
attention as potential sources to produce therapeutic agents, food, cosmetics, energy,
and pharmaceuticals (Bule et al. 2018). The global market for nutraceuticals and
food supplements made from microalgae is well-established, increasing by five-fold
since the beginning of the century and has significant growth potential (Enzing et al.
2014). An estimated US$6.5 billion is the market value of microalgae worldwide,
of which US$2.5 billion comes from the health food industry, US$1.5 billion from
docosahexaenoic acid (DHA) manufacturing, and US$700 million from aquaculture.

DOI: 10.1201/9781003455950-1 1
2 Algae Refinery

About 7.5 million tonnes of microalgae biomass are produced annually throughout
the globe (Mobin and Alam 2017).
For the manufacturing of human nutrition and food supplements, thousands of
production systems are under operation (Benemann 2009). The high-value-added
compounds (HVACs) of microalgae may provide several health advantages, includ-
ing anti-inflammatory, antioxidant, antiaging, antibacterial, anti-obesity, and anti-
cancer properties, according to published data. They can be used to manufacture
medications, cosmeceuticals, and next-generation nutritional supplements (Goswami
et al. 2022a). Nowadays, microalgae are being utilized to produce: (i) high-value sub-
stances such as PUFAs, pigments, and phycobiliproteins (Mehariya et al. 2021a); (ii)
entire biomass used in foods, feeds, and nutraceuticals (Mehariya et al. 2021b); (iii)
production of biofuel and biofertilizers from processed biomass; or (iv) bioremedia-
tion by utilizing living microalgae (Cardenas et al. 2018).
In the current situation, only a certain microalgal component is used to produce
biofuels such as lipids and carbohydrates (Mehariya et al. 2022). A biorefinery strat-
egy is needed to utilize all the fractions of biomass to get through these limitations
and optimization of economics (Zhu et al. 2015). By combining bioprocessing and
chemical methods, bio-refineries produce biofuels as well as valuable products from
microalgal biomasses which have a lesser impact on the environment (Rizwan et al.
2018). To advance microalgae on the market, a biorefinery strategy is essential. The
biorefinery strategy has drawn the attention of researchers who are keen to make use
of the valuable components synthesized by microalgae (Zhuang et al. 2020).
In addition to all the beneficial compounds, they also assist in lowering atmo-
spheric CO2 and bioremediation. Over the past 40 years, numerous studies have been
conducted on microalgae-based bioremediation to remove contaminants from waste-
water (Gill et al. 2013). Similarly, it has been discovered that utilizing microalgae
biomass as biosorbents to remove heavy metals is a cost-efficient, environmentally
friendly, and efficient approach to do it (Leong and Chang 2020). By utilizing waste-
water and CO2 sequestration, microalgae cultivation connects two major areas of
concern: global warming and water pollution control/water recycling (Patel et al.
2017). These advantages make microalgae a potential candidate for the biorefinery
approach (Koyande et al. 2019).
The chapter briefly includes the classification and overview of reported microal-
gae. Moreover, it elaborates on the microalgal upstream and downstream biorefinery
systems such as cultivation and processing, Moreover, this chapter also elaborates on
main commercial products obtained from microalgal biomass and their sustainable
applications.

1.2 CLASSIFICATION AND OVERVIEW OF THE MICROALGAE

Microalgae represents a wide group of organisms; they are found in a variety of
sizes, shapes, and habitats. Microalgae are mainly classified on the basis of their
structural diversity, sizes, pigments, habitats, and nature of stored biomolecules
(Table 1.1) (Correa et al. 2017).

TABLE 1.1
Brief Classifications and Overview of Commercially Important Microalgae and Their Reported Products.
Sr. No. Division Structural features
1 Chlorophyta Cup-shaped chloroplast,
contain chlorophyll a
and b in a single
chloroplast.
2 Rhodophyta Cells are spherical, contain
a large single chloroplast
with a single central
pyrenoid, pigments found
in Rhodophyta are
chlorophyll a, d, and
phycoerythrin.
3 Haptophyta One or two pyrenoid-
containing chloroplasts,
antapical nucleus, possess
a haptonema.
4 Stramenopiles Typically have two
flagella, two rows of
tripartite hairs found on
flagella, plastid contains
four outer membranes.
5 Dinophyta Dinokont flagellation,
cells might be spherical
or oval, motile or not.
Introduction to Microalgae and Its Refinery
Habitat Major commercial Microalgal species References
products
Inhabit in freshwater, Astaxanthin, β-carotene, Dunaliella salina, Heimann and
marine, or perhaps in protein, fatty acids Haematococcus pluvialis, Huerlimann
terrestrial Chlorella vulgaris, (2015); Goswami
environments. Chlamydomonas sp. et al. (2022a),
Shah et al. (2016)
Mostly marine, Phycobilin, pigments, Porphyridium cruentum, Román et al.
freshwater species cosmetics, antioxidants Rhodella reticulata (2002); Chen
are rare. et al. (2010)
Mostly marine As a feed in Pavlova salina, Isochrysis Jordan (2012)
aquaculture, fatty acids galbana
Mostly in Marine water Aquaculture feed, Nannochloropsis oculata, Heimann and
and some species are protein, laminarin Chaetoceros muelleri, Huerlimann
terrestrial. Skeletonema costatum (2015); Blanfuné
et al. (2016);
Chen et al. (2021)
Mainly marine, with a DHA, pigments, Crypthecodinium cohnii, D’Alessandro et al.
few freshwater species, antioxidants Gymnodinium sp. (2016); Schagerl
photosynthetic, 50% et al. (2003)
3
are heterotrophs.
4 Algae Refinery

1.3 UPSTREAM AND DOWNSTREAM

PROCESSING OF MICROALGAE
1.3.1 Microalgae Cultivation
Microalgae cultivation for the production of value-added products has received a lot
of attention in the past few decades. To effectively improve microalgae productivity
optimization of growth conditions and selection of appropriate cultivation method
are crucial. Various types of cultivation systems and modes used to cultivate microal-
gae are mentioned in the subsequent subsections.

1.3.1.1 Types of Cultivations Systems

There are two main cultivation systems such as open systems (circular pond and
raceway pond) and closed systems (photobioreactors), which are used to cultivate the
microalgae both in the laboratory or commercial scale (Figure 1.1a).

(a) Open systems: In open systems, raceway ponds, open ponds, circular ponds,
and unstirred ponds are generally used for large-scale cultivation (Mehariya
et al. 2021c). Microalgae production in open ponds is perhaps the oldest and
most straightforward scientific approach for cultivating algae (Jerney and
Spilling 2020). Compared to closed approaches or photobioreactors, open
cultivation methods have much lower production costs. They are compara-
tively easy to operate and scale up (Kusmayadi et al. 2020). However, there

FIGURE 1.1 General representation of types of (a) microalgae cultivation systems and

(b) types of microalgae cultivation modes.
Introduction to Microalgae and Its Refinery 5

are certain drawbacks in addition to these benefits, as open systems need a

lot of space to scale up and are vulnerable to contamination and bad weather
(Narala et al. 2016)
(b) Closed systems: The term “closed systems” refers to photobioreactors,
which do not directly exchange gases or other contaminants with the out-
side environment of the cultivation systems (Yen et al. 2019). Closed pho-
tobioreactors provide benefits including improved cultivation parameter
control and a lower chance of contamination (Apel and Weuster-Botz 2015).
Photobioreactors (PBRs) are far more capable of supporting much higher
photosynthesis effectiveness, biomass productivity, and biomass concentra-
tion than open systems (Wang et al. 2012). However, higher operational and
capital expenses are still big challenges in close-system cultivation (Sirohi
et al. 2022).

1.3.1.2 Modes of Cultivations

There are three major types of cultivation modes, that is, (a) photoautotrophic, (b)
heterotrophic, and (c) mixotrophic (Figure 1.1b).

(a) Photoautotrophic cultivation: It is the most common and natural mode

of cultivation where energy requirement is fulfilled by sunlight/LED and
atmospheric CO2 utilized as a carbon source (Zhan, et al. 2017).
(b) Heterotrophic cultivation: In this mode, organic compounds are supplied in
a bioreactor that fulfills both energy and carbon requirement in a dark con-
dition (Wang et al. 2014).
(c) Mixotrophic cultivation: Inorganic carbon (CO2) and organic compounds
are both utilized as carbon sources, and sunlight is utilized as an energy
source (Abreu et al. 2012). This is considered as the best method for the
treatment of wastewater (Cicco et al. 2021b).

1.3.1.3 Growth Parameters’ Optimizations

Optimization of growth parameters such as light, temperature, carbon source, and
pH of the growth medium can maximize the production of algal biomass.

(a) Light: It is required as an energy source for the process of photosynthesis.

The light to the algae culture is provided through sunlight or artificially
using light emitting diode (Wang et al. 2014). Optimum light intensity varies
from species to species. However, 3,000–5,000 lux intensity is considered
optimum for most of the microalgae (Goswami et al. 2022b). Day–night
cycle is also an optimal for the growth of microalgae because continued
illumination can lead to the photoinhibitions. It is suggested that contin-
uous light promotes the growth of microalgae (Cicco et al. 2021a). While
Goswami et al. (2023) reported that 12:12 hour day–night cycles promote
the growth of Tetraselmis indica.
(b) Temperature: It shows a positive correlation with microalgal growth, but
high temperature leads to oxidative stress (Esther et al. 2017). A temperature
range of 20–35℃ is considered optimum for most microalgae (Brindhadevi
6 Algae Refinery

et al. 2021). However, some strains also survived into the low temperatures
(15℃) (Goswami et al. 2022b, 2022c).
(c) pH is a key factor that limits the activity of enzymes that are involved in
proteins and pigment synthesis pathways (Qiu et al. 2017). Slightly alka�-
line (7–9) pH is favorable for most of the microalgae (Hossain and Mahlia
2019). Moreover, it also facilitates the availability and uptake of nutrients to
microalgae and promotes their growth (Goswami et al. 2023).
(d) Carbon source is a major component of algal biomass and its coproducts.
Therefore, the optimization of carbon sources significantly influences algal
biomass production. Both CO2 and organic compounds (i.e., glucose, glyc-
erol, sodium acetate, and citric acids) can be utilized as carbon sources
(Zhan, et al. 2017; Goswami et al. 2022b, 2022c).

1.3.2 Downstream Processing

The downstream processing of microalgae consists of three steps: harvesting, dry-
ing, and extraction of biomolecules and conversion into different HVAC products
(Figure 1.2) (Yew et al. 2019; Gu et al. 2022). Microalgae biomass was utilized for
the generations of pigments and carotenoids. While lipids and fatty acid are con-
verted into biofuels products, but production of biofuel from microalgal biomass is
not commercialized yet (Goswami et al. 2022d).

1.3.2.1 Harvesting
The recovery of the cultivated microalgal biomass from their growth medium is
referred to as harvesting. The ideal method for the recovery of high biomass at a
low cost requires a small amount of energy, maintenance, and operational costs
(Khoo et al. 2020). Centrifugation, flocculation, filtration, flotation, magnetic sep-
aration, electrolysis, ultrasound, and immobilization are common methods used
for algal biomass harvesting (Kim et al. 2013). However, yet, no efficient harvest-
ing methods are discovered. Many scientific investigations are carried out to find
low-cost harvesting techniques. Among them bioflocculation-based cultivations
are cheap, reliable, and energy-extensive process compared to others (Goswami
et al. 2022e).

FIGURE 1.2 Upstream cultivation and downstream processing of microalgae biomass.

Introduction to Microalgae and Its Refinery 7

1.3.2.2 Drying
Dewatering/drying is a key process in downstream processing in which water
is removed from the harvested cell culture to obtain biomass paste (Guldhe et al.
2014). Solar drying, convective drying, spray drying, and freeze drying are common
methods of biomass drying. However, the selection of a suitable drying method is
extremely dependent on the properties of the microalgal biomass (e.g., cell size, sur-
face charge, salt concentration, pH) (Chen et al. 2015).

1.3.2.3 Extraction
The extraction process is crucial for recovering useful compounds from microalgae.
Most of the by-products are encapsulated in microalgae cells (Salinas-Salazar et al.
2019). Therefore, the cell wall must be disrupted for the release of the desired mol-
ecules (Piasecka et al. 2014). Ultrasound extraction, enzymatic lysis, and chemical
and mechanical techniques are some common examples of extraction procedures.
Mechanical methods are utilized for cell rupture in large-scale applications because
they provide full cell destruction with a high output yield (Onumaegbu et al. 2018).
However, mechanical methods are more energy-intensive processes and increase
the cost of extractions. Enzyme-assisted extraction through mono-based enzymes,
such as cellulase, lipases, mannase, or cocktail enzymes, can lead to the cheapest
methods of biomolecules’ extraction from biomass. However, it is a time-consum-
ing process as compared to other methods. While green solvent-based extraction or
supercritical CO2-based extraction was far better and cheapest, greener methods for
the extractions of biomolecules (Goswami et al. 2021b).

1.4 ALGAE BIOREFINERY AND APPLICATIONS

OF ITS PRODUCTS
1.4.1 Bioenergy Products
Fuels produced through algal biomass are a sustainable, possibly environmentally
friendly, and affordable alternative source. Therefore, microalgae became a promis-
ing candidate to produce bioenergy.

1.4.1.1 Biodiesel
Biodiesel is a mono-alkyl ester that may be utilized in normal diesel engines with
little-to-no modification. It is generated by transesterifying triglycerides or natu-
ral fatty acids using short-chain alcohols. Experimental evidence suggests that
biodiesel is less harmful to the environment than petro-diesel, as it produces less
gaseous pollutants overall and no net CO2 or sulfur emissions (Rawat et al. 2013).
Microalgae biomass has great potential to be utilized as feedstock for biodiesel
compared to other vegetables oil because microalgae can multiply very fast and
contain a significant amount of oil in their biomass. Usually, microalgae double
their biomass within 24 hours, and oil content can reach up to 80% of their dry
biomass weight. These features make microalgae a potential candidate for bio-
diesel production (Chisti 2007). The diatom Phaeodactylum tricornutum, several
8 Algae Refinery

Chlorella species, many strains and species of the eustigmatofite, Nannochloropsis,

Chlamydomonas biomass are the most promising sources for biodiesel production
(Gouveia et al. 2017). Four main processes required for biodiesel production from
microalgae are (i) cultivation, (ii) harvesting, (iii) oil extraction, and (iv) transester-
ification (Wahlen et al. 2013).
However, there are still many difficulties associated with producing biodiesel from
algal biomass. According to the latest data, producing biodiesel from microalgal
biomass is more expensive than fossil fuels (Chowdhury and Loganathan 2019).
Currently, various efforts are underway to find the most productive algae species and
the generation of an in-situ transesterification directly from the biomass to reduce the
time and cost of biodiesel production (Nwokoagbara et al. 2015).

1.4.1.2 Bioethanol
At present, ethanol is considered the most popular biofuel which can be generated
from microalgal biomass. Utilizing microalgal biomass to produce ethanol offers
benefits because microalgal starch and cellulose are similar to higher plant starch,
and compared to agricultural crops, microalgal biomass has a low lignin content,
which makes the pretreatment process quite easy (Ho et al. 2013). Therefore, it is
recognized as an excellent raw material for the production of bioethanol (Maia
et al. 2020). By employing chemical or enzymatic methods, these polysaccharides
can be hydrolyzed to produce monosaccharides, and they can be fermented to get
bioethanol. With their high carbohydrate content, species from the genera Chlorella,
Chlamydomonas, Scenedesmus, Chlorococcum, and Tetraselmis represent ideal can-
didates for the production of bioethanol (Simas-Rodrigues et al. 2015). The process
required for bioethanol production primarily depends on the nature of algal biomass.
The process primarily consists of upstream and downstream activities that include
cultivation, harvesting, pretreatment, saccharification, fermentation, and product
recovery (Harun and Danquah 2011). The release and availability of fermentable
sugars for the fermentation process are dependent on biomass pretreatment, which is
a critical step (Ho et al. 2013).
However, like any other industrial process, the economics of algal biomass-based
bioethanol production must be considered before it can be commercialized. The pro-
cess of producing algal-based bioethanol faces several challenges such as the cost of
pretreatment and the degradation of sugars during hydrolysis, which must be solved
before it can be produced successfully on a large scale and for commercial use (Li
et al. 2014). There are currently several attempts in the process to boost bioethanol
production yields; including raising biomass growth rates, altering culture conditions
to produce more carbohydrates, or enhancing the efficiency of converting carbohy-
drates to ethanol (Lakatos et al. 2019) and leftover biomass could be used for bio-
polymer production (Mehariya et al. 2023).

1.4.1.3 Biomethane
Currently, the primary methods for producing biofuel from microalgae biomass
include extraction, hydrothermal liquefaction, pyrolysis, and anaerobic digestion.
Notably, the dewatering of the microalgae slurry before the extraction, hydrothermal
Introduction to Microalgae and Its Refinery 9

liquefaction, and pyrolysis procedures makes these conversion methods which are
energy-expensive processes. Additionally, only lipids can be extracted during the
extraction process, which leads to the wasting of proteins and carbohydrates (Xiao
et al. 2019). Typically, extremely high temperatures or pressures are utilized in the
hydrothermal liquefaction and pyrolysis processes, which require complicated sys-
tems and expensive operating costs (Wang et al. 2017). Anaerobic digestion can
utilize wet biomass directly to generate methane, saving the energy needed for dewa-
tering and lowering the overall utilization of energy in biogas generation; in addition,
it is a carbon-neutral approach (Damtie et al. 2021).
The following four steps are involved in the production of biomethane via
anaerobic digestion: (i) hydrolysis of biomass, (ii) acidogenesis, (iii) acetogene-
sis, and (iv) methanogenesis. The hydrolysis process involves the biodegradation
of the polymeric substances of the microalgal biomass, such as proteins, lipids,
and carbohydrates, by the specific microorganisms in the digester, enabling the
production of simple molecules like fatty acids, glucose, and amino acids. In the
acidogenic phase, simple molecules are transformed into small fatty acids and
molecules into gaseous form such as CO2, bio-H2, and CH3COOH. By acetogen-
esis, acetogenic bacteria transform fatty acids into acetic acid and H 2, which ulti-
mately metabolize into methane by the action of methanogenic bacteria in the
methanogenesis step (Zabed et al. 2019; Prajapati and Malik 2015). Chlorella,
Tetraselmis, Spirulina, and Scenedesmus, are ideal microalgal genera to produce
biomethane (González-Fernández et al. 2012). However, some limitations of bio-
methane generation put a constraint on it from being feasible on a large scale.
The major challenges are that anaerobic digesters have a slow biodegradation rate
and low C/N proportions in a digester due to a large concentration of proteins
(Lu et al. 2019; Passos et al. 2018).

1.4.1.4 Biohydrogen
Currently, the majority of industrial H2 production technologies are based on ther-
mochemical processes, which typically require significant fossil fuel usage. Reports
suggest that producing biohydrogen from microalgae is an extremely attractive
approach to achieving zero carbon emissions and bioenergy sustainability (Li et al.
2022). With water as a by-product, hydrogen demonstrates the significant benefit of
CO2-free combustion (Limongi et al. 2021).
Microalgae produce biohydrogen via two majors procedures: (i) light-dependent
pathway and (ii) light-independent pathway. The light-dependent pathway is known
as biophotolysis in which the action of light energy causes the split of the H2O into
H2 and O2. The light-independent pathway includes dark fermentation of microalgal
biomass, which is carried out by anaerobic bacteria (Singh et al. 2022; Goswami
et al. 2021a). Chlorella, Chlamydomonas Scenedesmus, and Tetraspora are consid-
ered ideal microalgal genera for dark fermentation (Khetkorn et al. 2017).
However, some drawbacks limit the commercial scaling of biohydrogen produc-
tion from microalgae such as the O2-sensitivity of enzymes, lower yield, lower rate
of biomass conversion, and the lack of understanding of the involved enzymes and
biochemical reactions (Goswami et al. 2021b).
10 Algae Refinery

1.4.2 Pharmaceuticals
The health industry has traditionally used secondary metabolites derived from plants.
Currently, researchers have switched to microalgae as a production platform, because
of the lower yields and seasonal changes of plant sources. Microalgae cells can pro-
duce novel chemical compounds that are believed to be challenging to obtain by
chemical synthesis. Microalgae are used for manufacturing many kinds of pharma-
ceutical products which are mentioned in the subsequent subsections (Jha et al. 2017).

1.4.2.1 Single-Cell Protein

Microalgae can be grown in controlled environments with little water consumption,
and they express high levels of protein. Numerous species of microalgae have amino
acid profiles that are similar to those of well-balanced proteins as described by WHO/
FAO (Janssen et al. 2022). Comprehensive research and nutritional studies have shown
that these algal proteins are superior to traditional vegetable proteins (Montenegro-
Herrera et al. 2022). Spirulina, Chlorella, Dunaliella, and Scenedesmus genus are
the most common microalgae used in the human diet and protein production at the
commercial scale (Barka and Blecker 2016).
However, in addition to its beneficial characteristics, microalgal single-cell pro-
tein has some drawbacks such as high nucleic acid amount in biomass, increasing
uric acid in serum that results in kidney stones formation, the cell wall of microal-
gae being nondigestible by humans, endotoxins, and high risk of contamination.
Selection of appropriate microalgae, suitable substrate, and optimization of condi-
tions may help to overcome these limitations (Sharif et al. 2021; Becker 2007).

1.4.2.2 Antiviral Agents

There are currently no effective vaccinations available against many common viral
diseases, and the manufacturing of vaccines against other viruses, such as HIV and
HCV, has shown to be ineffective (Mahendran et al. 2021). Some antiviral substances
can be found naturally, and microalgae are one of the most promising sources for
their synthesis. One of the earliest research studies on the antiviral action of microal-
gae was carried out on Chlorella pyrenoidosa and was found to have an inhibitory
impact on mice against the vesicular stomatitis virus (VSV) (Carbone et al. 2021).
Santoyo et al. (2010) studied the antiviral effect of ethanol extract of Chlorella vul-
garis against herpes simplex virus type 1 and found significant inhibition of infec-
tion. Huleihel et al. (2001) studied the antiviral activity of red algae Porphyridium
sp. against Herpes simplex viruses and Varicella zoster virus and reported 50% inhi-
bition of infection.
Compounds produced by microalgae such as phycobiliprotein, fucoidan, xylo-
mannan, and polyphenol directly inactivates viruses by binding them irreversibly,
stopping them from interacting with their target cells or destroying the virion’s struc-
tural integrity (Reynolds et al. 2021). Genus Chlorella, Dunaliella, Chlamydomonas,
and Scenedesmus are the most popular microalgae that contain antiviral substances
and show a significant inhibition of viral infections (Khavari et al. 2021).
Introduction to Microalgae and Its Refinery 11

1.4.2.3 Antibacterial Agents

An important issue that has raised the demand for new antibacterial chemicals is
the growth of bacteria that are more resistant to antibiotics. Metabolites synthe-
sized by microalgae such as phenolics, terpenes, glycolipids, fatty acids, glycolip-
ids, and alkaloids have antimicrobial properties. Therefore, several investigations
are focused on the antibacterial potential of green microalgae (Little et al. 2021).
Bhuyar et al. (2020) investigated the antibacterial activity of Oscillatoria extract
against Gram –ve bacteria Streptomyces aureus, Bacillus subtilis, and Gram +ve
bacteria Escherichia coli, and the results show significant inhibition of bacterial
growth. Hidhayati et al. (2022) investigated the antibacterial activity of ethyl ace-
tate extract of Spirulina platensis against Propionibacterium, and Enterobavter
aerogenes result shows that the extract has strong antibacterial properties against
bacteria. Ethanolic extract of Chlorella vulgaris contains antibacterial compounds:
38.8% linoelaidic acid and 30.0% phytol that show strong antibacterial activity
against Escherichia coli (Kim et al. 2017). Ethanol extract of Chlorella sp. and
Scenedesmus sp., methanol extract of Chlorococcum and Tetraselmis, and ethyl
acetate extract of Nannochloropsis show effectiveness against bacterial growth
(Ognistaia et al. 2022).

1.4.2.4 Antioxidants
In past decades, scientists have been searching for a natural replacement for syn-
thetic antioxidants; microalgae are considered a potential source of antioxidants
because they stimulate antioxidant defense against oxidative stress and prevent cells
from reactive oxygen species (Assunção et al. 2017). Carotenoids, pigments, and
fatty acids produced by microalgae contain antioxidant properties (Gauthier et al.
2020). Microalgae Chlorella, Tetraselmis, Phaeodactylum, and Bot ryococcus con-
tain large amounts of antioxidants. Thus, they are considered a potential source of
antioxidants (Goiris et al. 2012). Ascorbic acid, carotenoids, glutathione, tocoph-
erols, and phenolic compounds are secondary metabolites of microalgae, and they
can neutralize reactive oxygen species (Coulombier et al. 2021). Guedes et al. (2013)
studied that Scenedesmus obliquus contains a significant amount of antioxidants
such as zeaxanthin, violaxanthin, neoxanthin, and luteoxanthin that protect DNA
from oxidative damage. Widowati et al. (2017) evaluated the antioxidant activity
of Dunaliella salina, Isochrysis galbana, and Tetraselmis chuii and found that
Isochrysis galbana shows excellent antioxidant potential followed by Dunaliella
salina. Bellahcen et al. (2020) carried out a comparison study of ethanolic, lipid,
and aqueous extracts of Spirulina platensis for their antioxidant activity, and result
shows that ethanolic extract has the highest antioxidant activity. Nasirian et al. (2018)
examined the effect of the antioxidant activity of Spirulina platensis on diabetes;
result shows that the activity of antioxidant enzymes was significantly increased and
lowered the level of diabetes. Antioxidants produced by microalgae show antican-
cer, anti-inflammatory, antidiabetic, neuroprotective, and cardiovascular protective
effects (Yuan et al. 2011).
12 Algae Refinery

1.4.3 Cosmetics
Cosmetics are products that improve the appearance of the skin (Mourelle et al.
2017). Synthetic cosmetics can have harmful effects on the skin. Due to the excellent
antioxidant property of microalgae by-products, they can be utilized to produce cos-
metics (Zhuang et al. 2022). Microalgal metabolites such as β-carotene, folic acid,
pantothenic acid, and vitamin B12 have antimicrobial, UV protective, and anti-aging
properties and can also repair and heal skin; due to these properties, microalgae draw
the significant attention of cosmeceutical industries (Yarkent et al. 2020). Dunaliella,
Chlorella, Arthrospira, and Haematococcus are considered potential microalgae to
produce cosmetics at a commercial scale (Morocho et al. 2020).

1.4.4 Chemicals
Microalgae can produce a wide variety of chemicals and metabolites (Figure 1.3),
and due to their photoautotrophic nature, they are considered sustainable and low-
cost alternatives to synthetic chemicals.

1.4.4.1 Pigments
Due to the harmful effects of synthetic colorants, natural pigments produced by
microalgae become attractive alternatives. Microalgae synthesize a wide variety

FIGURE 1.3 Microalgae biomass-based biorefineries and different high value-added products.
Introduction to Microalgae and Its Refinery 13

of pigments such as chlorophyll a, b, and c; xanthophylls; phycobiliproteins;

astaxanthin; and β-carotene (Begum et al. 2016). Due to their antioxidant nature,
these pigments receive significant value in the world food market (Sun et al. 2023).
Natural pigments are also utilized in the production of cosmetics due to their
anti-aging property (Morocho et al. 2020). Chlorella, Haematococcus, Spirulina,
and Dunaliella are the most studied microalgae to produce pigments (Silva et al.
2020). However, the usage of these pigments still faces numerous obstacles such
as the expensive production costs and instability of isolated pigments (Pagels
et al. 2020).

1.4.4.2 Biopolymers and Bioplastics

Due to its non-degradability and generation of waste in huge amounts, synthetic
plastic places harmful effects on the environment (Mastropetros et al. 2022).
Biopolymers are considered sustainable alternatives to fossil-based synthetic
polymers due to their biodegradability and renewability (Madadi et al. 2021).
Microalgae synthesize poly-hydroxy-alkanoate (PHA) and starch used for bio-
polymer synthesis, and characteristics of these polymers are similar to those of
petrochemical polymers, which safe for the environment (Rajpoot et al. 2022).
Biopolymers are produced by the fermentation of microalgal biomass or by com-
bining biomass by mixing some additives (Onen et al. 2020). Spirulina, Chlorella,
Neochloris, and Phaeodactylum are the potential microalgae for the synthesis of
biopolymers due to their starch or polyhydroxy acetone (PHA)-rich nature (Onen
et al. 2020).

1.4.4.3 Nanoparticles
In recent decades, the usage of nanoparticles has significantly increased due to
their wide application in the medical and pharma sectors. Due to harmful effects
on the environment, physical and chemical methods are not considered sustainable
for the biofabrication of nanoparticles (Jena et al. 2013). Microalgae take in metals
ions during detoxification so they are considered a novel alternative to physical and
chemical methods and also considered potential candidates for wastewater treatment
(Mohseniazar et al. 2011; Agarwal et al. 2019). Zayadi et al. (2020) synthesized gold
nanoparticles using Chlorella and Spirulina which resulted in higher stability and
less reactivity of gold nanoparticles. Ebrahiminezhad et al. (2016) synthesized silver
nanoparticles by utilizing Chlorella vulgaris that shows anticancer and antioxidant
properties. Muthusamy et al. (2017) synthesized silver nanoparticles by utilizing
Spirulina platensis that shows antibacterial activity against Staphylococcus and
Klebsiella.

1.4.5 Food products

Protein, carbohydrates, lipids, and other physiologically active substances can be
synthesized by microalgae in large amounts (Table 1.2). In this scenario, microalgae
are becoming more and more popular in food applications (Rahman 2020).
14 Algae Refinery

TABLE 1.2
Protein, Lipid, and Carbohydrate Composition of Commercially Important
Microalgae.
Microalgae Protein (%) Lipid (%) Carbohydrate (%) References
Chlorella vulgaris 47.82 ± 0.05 13.32 ± 0.07 8.08 ± 0.09 Tokuşoglu and Üunal
(2003)
Isochrysis galbana 37 24 11 Reiriz et al. (1989)
Tetraselmis gracilis 33.02 ± 0.43 7.95 ± 0.42 29.96 ± 1.99 Gorgonio et al. (2013)
Porphyridium 34.1± 4.4 6.53 ± 0.46 32.1 ± 5.6 Fuentes et al. (2000)
cruentum
Dunaliella salina 25.67 18.02 40.21 Muhaemin and
Kaswadji (2010)
Spirulina platensis 63 7.53 15.35 Tokuşoglu and Üunal
(2003)
Nannochloropsis sp. 28.8 18.4 35.9 Rebolloso et al. (2001a)
Phaeodactylum 36.4 18 26.1 Rebolloso et al.
tricornutum (2001b)
Galdieria 32.5 1.77 62.9 Graziani et al. (2013)
sulphuraria
Tetraselmis indica n.d. 44 ± 0.47 n.d. Goswami et al. (2023)

1.4.5.1 Fatty Acid

Polyunsaturated fatty acids (PUFAs) are not synthesized by the human body so they
are known as essential fatty acids. Nowadays, PUFAs are obtained from fish oil, but
their application in food additives is limited due to their bad smell, taste, and poor
oxidative stability (Sayeda et al. 2015). In contrast, PUFAs obtained from microalgae
have good taste and show medicinal benefits such as anticancer, anti-obesity, anti-
diabetic properties; reduction in inflammation; and prevention from cardiovascular
diseases, and so they are considered the potential alternative to fish and vegetable
oil (Kumar et al. 2019). Microalgae comprise significant amounts of PUFAs such as
eicosapentaenoic acid (EPA), linolenic acid (ALA), and DHA (Santos-Sánchez et al.
2016; Mehariya et al. 2021b).
Currently, microalgae species from the genera Chlorella, Isochrysis, Spirulina,
Haematococcus, Dunaliella, Nostoc, Nannocloropsis, Porphyridium, Arthrospira,
Schizochytrium, and Thalassioi are used for the production of fatty acids at commer-
cial scale (Maltsev and Maltseva 2021).

1.4.5.2 Protein and Amino Acids

Protein is an essential component of the human diet because it provides the majority
of the required nitrogen and essential amino acids to the human body (Torres-Tiji
et al. 2020). Due to its higher nutritional property, the presence of essential amino
acids, and being low allergic as compared to milk and soy protein, microalgal pro-
tein is considered a novel protein supplement (Soto-Sierra et al. 2018). Due to higher
Introduction to Microalgae and Its Refinery 15

protein content (55–70%), various microalgae such as Dualiella, Chlorella, and

Spirulina are considered potential sources for the production of protein supplements.
It is common and well-established fact to consume whole microalgae biomass in
food and feed (Amorim et al. 2021). Nowadays, Dunaliella and Sprulina are directly
sold as protein supplements, aquafeed, or animal feed only after drying without any
processing (Matos 2019).

1.4.5.3 Carbohydrates
Microalgae are photoautotrophic microorganisms that convert atmospheric CO2 into
carbohydrates, where synthesized carbohydrates can be stored inside cells or may act
as a structural component (Markou et al. 2012). Structural carbohydrates of microal-
gae do not contain lignin, so they are considered superior animal feed and substrate
for bioethanol production as compared to plant-based biomass (Debnath et al. 2021).
Microalgae with higher carbohydrate content are considered a potential approach for
biomethane and biohydrogen production (Liu et al. 2012).
Chlamydomonas, Dunaliella, Scenedesmus, Nannochloropsis, and Tetraselmis
can produce more than 50% carbohydrate under favorable conditions, so they are
considered potential microalgae for commercialization (Spolaore et al. 2006).

1.4.6 Environmental Applications

1.4.6.1 Carbon Dioxide Sequestration
The atmospheric CO2 level is rising day by day due to anthropogenic activities or the
burning of fossil fuels. CO2 is a major greenhouse gas that contributes to more than
half of the greenhouse effects (Singh and Ahluwalia 2013). Due to rapid growth,
higher photosynthetic efficiency, and wide environmental adaptability, microal-
gae-based carbon sequestration is considered the most effective, economical, and
sustainable method to overcome the greenhouse effect (Xu et al. 2019). More than
25% of annual anthropogenic CO2 is absorbed by oceans, converted into carbonic
acid, and this causes acidification of the ocean; algae can take in carbonate via the
carbon concentration mechanism (CCM) and prevent the ocean from acidification
(Devi et al. 2013). Simultaneously, produced algal biomass is utilized in the produc-
tion of biofuel, pharmaceuticals, cosmetics, nutraceuticals, pigments, and various
other valuable products (Banerjee et al. 2020; Goswami et al. 2022c).

1.4.6.2 Wastewater Treatment

Word population is rising day by day that is accelerating urbanization, and urban-
ization leads to wastewater generation. Consumption of wastewater cause several
health-related issues such as diarrhea, vomiting, and endocrine disruption (Goswami
et al. 2022d). Therefore, wastewater treatment becomes essential to recycle it and
resolve the problem of water shortage (Singh et al. 2022). Among various wastewa-
ter treatment techniques, microalgae-based treatment received much attention due
to its low cultivation cost, sustainable nature, and higher nutrient removal capacity
(Goswami et al. 2022e, 2022f).
Microalgae could potentially be able to significantly reduce the number of nutri-
ents in wastewater, and treated wastewater fulfills the strict guidelines for discharge
16 Algae Refinery

and reuse of the water (Li et al. 2019; Iovinella et al. 2022). Microalgae can effi-
ciently utilize nitrogen and phosphorus, as well as organic and inorganic carbon
from wastewater, and harvested biomass can be utilized in manufacturing several
HVAC products (Mohsenpour et al. 2021). Microalgae can take in heavy metals
from wastewater, which can be utilized in the production of nanoparticles (Goswami
et al. 2022f). Based on these advantages, microalgae-based wastewater treatment is
considered a potential alternative to conventional chemical-based treatment methods
(Nagarajan et al. 2019).

1.5 FUTURE PROSPECTS AND CONCLUSIONS

Microalgae-based refineries make complete use of biomass while lowering produc-
tion costs and environmental pollution. Microalgae have a huge potential as raw
material for biorefinery applications because they contain significant amounts of bio-
molecules that have various energy, health, and industrial applications. In addition,
the cultivation of microalgae can remediate wastewater and reduce the CO2 from the
atmosphere. However, due to high cultivation and harvesting costs (20–30% of total
production cost) and low light penetration in phototrophic and contamination in het-
erotrophic cultivation, the industrial production of microalgal by-products is not yet
cost-effective. Moreover, biomass cultivated in polluted water is not considered safe
for the production of pharmaceuticals and food products. Therefore, the commer-
cial application of microalgae is limited only to biofuel production, so there was an
immediate need to integrate it with other valuable by-product production. The imple-
mentation of the macroalgal biorefinery is crucial to support a microalgae-based
economy. Research on the genetic modification of microalgae strains to face harsh
environmental conditions is still needed in upcoming future.

ACKNOWLEDGMENTS
Authors did not receive any specific grant from funding agencies in the public, com-
mercial, or not-for-profit sectors.

CONTRIBUTIONS
Udaypal has contributed toward investigation, resources, and roles/writing the orig-
inal draft; Rahul Kumar Goswami toward review and editing; Sanjeet Mehariya
toward review and editing; and Pradeep Verma has contributed to conceptualiza-
tion, editing, and supervision.

REFERENCES
Abreu, Ana Paula, Bruno Fernandes, António Augusto Vicente, José Teixeira, and Giuliano
Dragone. 2012. “Mixotrophic cultivation of Chlorella vulgaris using industrial dairy
waste as organic carbon source.” Bioresource Technology 118: 61–66. https://doi.
org/10.1016/j.biortech.2012.05.055.
Agarwal, Prashant, Ritika Gupta, and Neeraj Agarwal. 2019. “Advances in synthesis and appli-
cations of microalgal nanoparticles for wastewater treatment.” Journal of Nanotechnol-
ogy 2019: 739271 3. https://doi.org/10.1155/2019/7392713.
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 Figure 79. De Konick’s Apparatus.

467. DeKonick’s Modification of Schloesing’s

Method.—This modification consists in an
arrangement of the gas delivery-tube, whereby the
regurgitation of the water in the measuring burette into
the evolution flask is prevented by a device for sealing
the delivery-tube with mercury.[299] The apparatus is
arranged as shown in Fig. 79. The flask in which the
Figure 80. decomposition takes place is provided with a long neck,
End of
Delivery-
into which a side tube is sealed and bent upwards,
Tube. carrying a small funnel attached to it by rubber tubing.
The piece of rubber tubing carries a pinch-cock, by
means of which the solution containing the nitrate and
hydrochloric acid can be introduced into the flask. The small gas
delivery-tube is arranged as shown in the figure, and carries at the
end next the burette a device shown in Fig. 80. The cork represented
in this device has radial notches cut in it, so as to permit of a free
communication between the water in the burette and in the
pneumatic trough. The open end of the burette, when the apparatus
is mounted ready for use, rests on the notched surface of the cork,
and the end of the delivery-tube is placed in the crystallizing dish
resting on the bottom of the pneumatic trough.
The end of the delivery-tube, as indicated, has fused onto it a
vertical tube open at both ends and six to seven centimeters in
length, and carrying the notched cork already described. The
crystallizing dish in the bottom of the pneumatic trough is filled with
mercury until the point of union of the delivery-tube with the vertical
end is sealed to the depth of a few millimeters. As the gas is evolved
it bubbles up through the mercury into the measuring tube and the
displaced water passes out through the notches in the cork. Should
any back pressure supervene the mercury at once rises in the
delivery-tube which is of such a length as to prevent its entrance into
the flask. The operation can then be carried on with absolute safety.
To make an estimation there are placed in the flask about forty
cubic centimeters of ferrous chlorid solution containing about 200
grams of iron to the liter, and also an equal volume of hydrochloric
acid of one and one-tenth specific gravity. The side tube is also filled
up to the funnel with the acid. The contents of the flask are boiled
until all air is expelled, which can be determined by holding a test-
tube filled with water over the end of the delivery-tube. The solution
containing the nitrate is next placed in the funnel, the pinch-cock
opened and the liquid allowed to run into the flask by means of the
partial vacuum produced by stopping the boiling and allowing the
mercury to rise in the delivery-tube. All the solution is washed into
the flask by successive rinsings of the funnel with hydrochloric acid,
being careful to allow no bubble of air to enter. The contents of the
flask are again raised to the boiling-point and the nitric oxid evolved
collected in the nitrometer. The solution examined should contain
enough nitrate to afford from sixty to eighty cubic centimeters of gas.
Without refilling the flask, from eight to nine determinations can be
made by regenerating the ferrous chlorid by treatment with zinc
chlorid. Care must be exercised not to add the zinc chlorid in excess,
otherwise ammonia and not nitric oxid will be produced. The side
tube and funnel must also be carefully freed from zinc chlorid by
washing with hydrochloric acid.
 468. Schmidt’s Process.—In the case of a water, or the aqueous
extract of a soil, according to the content of nitric acid, from fifty to
one hundred cubic centimeters are evaporated to thirty cubic
centimeters, and the residue sucked into the generating flask of the
apparatus, Fig. 81, and, with the rinsings with distilled water,
evaporated again to from twenty to thirty cubic centimeters, and the
flask then connected, as shown in the figure, to a Schliff measuring
apparatus, B.[300] This apparatus is previously filled to i with
mercury, and the bulb g connected with k by a rubber tube.
The apparatus is then
filled with a twenty per
cent, previously boiled
and still warm, caustic
soda solution until the
bulb g is partially filled
when raised a little above
the cock h. Then h is
closed and g held, by an
appropriate support, on
about the same level with
h. The cock at b is then
closed and e opened.
Meanwhile the ebullition
in the flask is continued,
and the air bubbles rising
in the Schliff apparatus
are removed, from time
Figure 81. Schmidt’s Apparatus.
to time, by carefully
opening h and raising g.
When bubbles no longer come over, the cock at e is closed and at b
opened, and the steam issuing at a is conducted through a mixture of
ferrous chlorid and strong hydrochloric acid to free it, as far as
possible, from air. When the contents of the flask have been
evaporated to about five cubic centimeters, b is closed and the lamp
at once removed.
By carefully opening b about ten cubic centimeters of a mixture of
ferrous chlorid and hydrochloric acid are allowed to enter the flask,
when b is closed and the flask slowly heated until the positive
pressure is restored. The pinch-cock e is then opened and the
contents of the flask evaporated nearly to dryness. The cock e is
again closed and the flame removed. Another quantity (fifteen cubic
centimeters) of ferrous chlorid and hydrochloric acid solution is
sucked into the flask and the process of distillation repeated,
whereby the whole of the nitric oxid is collected in h. The nitric oxid
evolved is measured in the usual way and calculated to nitric acid,
one cubic centimeter of nitrogen dioxid being equal to 2.417
milligrams of nitric acid.
469. Merits of the Ferrous Chlorid Process.—The possibility
of an accurate determination of nitrates; by decomposition with a
ferrous salt in presence of an excess of acid, has been established by
many years of experience and by the testimony of many analysts. The
method is applicable especially where the quantity of nitrate is not
too small and when organic matter is present. In the case of minute
quantities of nitrate, however, the process is inapplicable and must
give way to some of the colorimetric methods to be hereafter
described.
In respect of the apparatus modern practice has led to the
preference of that form which does not require the use of carbon
dioxid for displacing the air. Steam appears to be quite as effective as
carbon dioxid and is much more easily employed. That form of
apparatus should be used which is the simplest in construction and
has the least cubical content.
The measurement of the evolved gas is most simply made by
collecting over lye in an azotometer, reading the volume, noting the
reading of the barometer and thermometer and then reducing to
standard conditions of pressure and temperature by the customary
calculations. Where a very strong lye is used the tension of the
aqueous vapor may be neglected. While every analyst should have a
thorough knowledge of the ferrous chlorid method and the principles
on which it is based it can not be compared in simplicity to the later
methods with pure nitrates which are based on the conversion of the
nitric acid into ammonia by the action of nascent hydrogen. In
accuracy, moreover, it does not appear to have any marked
advantage over the reduction methods.
470. Mercury and Sulfuric Acid Method.—This simple and
accurate method of determining nitric acid in the absence of organic
matter is known as the Crum-Frankland process.[301]
The method rests on the principle of converting nitric acid into
nitric oxid by the action of mercury in the presence of sulfuric acid.
The operation as at first described is conducted in a glass jar eight
inches long by one and a half inches in diameter filled with mercury
and inverted in a trough containing the same liquid. The nitrate to be
examined, in a solid form, is passed into the tube together with three
cubic centimeters of water and five of sulfuric acid. With occasional
shaking, two hours are allowed for the disengagement of the gas,
which is then measured.
471. Warington’s Modification.—A graduated shaking tube is
employed which allows the nitrate solution and oil of vitriol to be
brought to a definite volume. The nitrate solution, with rinsings, is
always two cubic centimeters and enough sulfuric acid is added to
increase the volume to five cubic centimeters. The sulfuric acid
should give no gas when shaken with distilled water. Any gas given
off in the apparatus before shaking, is not expelled but is included in
the final result. The persistent froth sometimes noticed where some
kinds of organic matter are present, is reduced by the addition of a
few drops of hot water through the stop-cock of the apparatus. The
nitric oxid is finally measured in Frankland’s modification of
Regnault’s apparatus.
This method, accurate for pure nitrates, unfortunately fails in the
presence of any considerable amount of organic matter.
According to Warington’s observations the presence of chlorids is
no hindrance to the accurate determination of both nitric and nitrous
acids by the mercury method. This simplifies the operation as carried
on by Frankland who directs that any chlorin present, be removed
before the determination of the nitric acid is commenced.
472. Noyes’ Method.—In the analyses made by Noyes for the
National Board of Health, the Crum-Frankland method was
employed.[302] The apparatus used was essentially that which is now
known as Lunge’s nitrometer and it will be described in the next
paragraph. No correction is made by Noyes for the tension of
aqueous vapor in the measurement of the nitric oxid because of the
moderate dilution of the sulfuric acid by the liquid holding the nitric
compounds in solution. The chlorin was not removed from the dry
residue of the evaporated water as its presence in moderate quantity
does not interfere with the accuracy of the process. In order to obtain
the amount of nitrogen in the form of nitrates, the total volume of
nitric oxid must be diminished by that due to nitrites present, which
must be determined in a separate analysis. The method of
manipulation is given in the following paragraph.
 473. Lunge’s Nitrometer.—The apparatus
employed by Noyes, in a somewhat more elaborate
form, is known as Lunge’s nitrometer.[303] This
apparatus is shown in Fig. 82. It consists of a burette,
a, divided into one-fifth cubic centimeters. At its
upper end it is expanded into a cup-shaped funnel
attached by a three-way glass stop-cock. Below, the
burette is joined to a plain tube, b, of similar size, by
means of rubber tubing. The apparatus is first filled
with mercury through the tube b, the stop-cock being
so adjusted as to allow the mercury to fill the cup at
the top of a. The cock is then turned until the mercury
in the cup flows out through the side tube carrying the
rubber tube and clamp. The three-way cock is closed,
and the solution containing the nitrate placed in the
cup. By lowering the tube b and opening the cock the
liquid is carefully passed into a, being careful to close
the cock before all the liquid has passed out of the
cup. By repeated rinsings with pure concentrated
sulfuric acid, every particle of the nitric compound is
finally introduced into a, together with a large excess
of sulfuric acid. The total volume of the introduced
liquid should not exceed ten cubic centimeters. The
Figure 82. Lunge’s Nitrometer. mixture of the mercury, nitric compound, and sulfuric
acid is effected by detaching a from its support,
compressing the rubber connection between a and b,
placing a nearly in a horizontal position, and quickly bringing it into a vertical position with
vigorous shaking.
After about five minutes the reaction is complete, and the level of the liquids in the two
tubes is so adjusted as to compensate for the difference in specific gravity between the acid
mixture in a and the mercury in b; in other words, the mercury column in b should stand
above the mercury column in a one-seventh of the length of the acid mixture in a. This
secures atmospheric pressure on the nitric oxid which has been collected in a. The measured
volume of nitric oxid should be reduced to 0° and 760 millimeters barometric pressure. Each
cubic centimeter of nitric oxid thus obtained corresponds to 1.343 milligrams NO; 2.417
milligrams N₂O₅; 4.521 milligrams KNO₃; 1.701 milligrams N₂O₃; 2.820 milligrams HNO₃;
and 3.805 milligrams NaNO₃.
474. Lunge’s Improved Apparatus.—Lunge has lately improved his apparatus for
generating and measuring gases and extended its applicability.[304] The part of it designed to
measure the volume of a gas is the same in all cases. For generating the gas, the apparatus
varies according to the character of the substance under examination.
The measuring apparatus is shown in Fig. 83. It is composed essentially of three tubes,
conveniently mounted on a wooden holder with a box base for securing any spilled mercury.
The support is not shown in the illustration.
The tubes A, B, C, are mutually connected by means of a three-way tube and rubber tubing
with very thick walls to safely hold the mercury without expansion. In the middle of the
measuring tube A, is found a bulb of seventy cubic centimeters capacity. Above and below the
bulb the tube is divided into tenths of a cubic centimeter, and its diameter is such, viz., 11.3
millimeters, that each cubic centimeter occupies a length of one centimeter. The upper end of
A is closed with a glass cock with two oblique perforations, by means of which
communication can be established at will, either through e with the apparatus for generating
the gas, or through d with the absorption apparatus, or the opening be completely closed.
The volume of air under the observed conditions which would
measure exactly 100 cubic centimeters at 0° and 760
millimeters pressure of mercury, is calculated by the formula
V = 100(273 + t)760
273(b − f) ;

where t equals observed temperature, b the barometric pressure

less the correction noted above and f the tension of the vapor of
water under existing conditions. For example:

Figure. 83.

Lunge’s Improved Apparatus.

Let the temperature be 18°

Barometric reading 755
Correction for t 2
Corrected barometer 753
Vapor of water tension 16

Then V = 100(273 + 18)760

273(753 − 16) = 109.9.

This indicates that 109.9 cubic centimeters of air would occupy a volume of 100 cubic
centimeters when subjected to standard conditions.
The tubes A, B, and C are filled with mercury of which about two and a half kilograms will
be required. By means of the leveling tube B, the stopper in C being opened, the mercury in C
is brought exactly to 109.9 cubic centimeters. The stopper in C is then closed, mercury
poured into D, which is then closed with a rubber stopper, carrying a small glass tube as
indicated in the figure.
 The leveling tube B serves to regulate the pressure on the gas in A and this is secured by
depressing or elevating it as the case may require.
The tube for reducing the volume to standard conditions of temperature and pressure, viz.,
0° and 760 millimeters of mercury, is shown in C. In its narrow part which has the same
internal diameter as A it is graduated into tenths of a cubic centimeter. The upper end of C is
furnished with a heavy glass neck D surmounted by a glass cup. In the neck is placed a
ground-glass stopper, carrying a groove below, which corresponds to a similar groove above
in the side of the neck whereby communication can be established at will between the interior
of C and the exterior. The joint is also sealed by pouring mercury into D as is shown in the
figure. When the stopper is well ground and greased the reduction tube may be raised or
lowered as much as may be necessary without any danger of escape or entrance of gas. To
determine the position of the reduction tube C the reading of the barometer and
thermometer at room temperature is taken. From the reading of the barometer subtract one
millimeter if the temperature be below 12°, two millimeters at a temperature from 12° to 19°,
three from 20° to 25°, and four above 25°.
When a gas has been introduced into the measuring tube A it is brought to the volume
which it would assume under standard conditions by adjusting the tube C in such a way as to
bring the level of mercury in C and A to the same point and the level of the mercury in C is
exactly at 100 cubic centimeters. The gas in A is then at the volume which it would occupy
under standard conditions and this volume can be directly read. This adjustment is secured
by moving the tubes B and C up or down. If gases are to be measured wet, a drop of water
should be put on the side of the upper part of C, and, if dry, of sulfuric acid, before the
adjustment for temperature and pressure.
475. Method of Manipulation.—By the action of mercury in the presence of sulfuric
acid, the nitrogen in nitrates, nitrites, nitrosulfates, nitroses, nitrocellulose, nitroglycerol, and
the greater number of explosives, may be obtained and measured as nitric oxid. The nitrogen
compounds are decomposed in the apparatus shown in Fig. 84.
To make an analysis, the apparatus is filled with mercury, through F, until the two
openings in the cock and i are entirely occupied with that liquid. The cock h is then closed,
and the nitrogen compound, in solution, introduced through g, care being taken that no air
enters g when F is depressed and h opened to admit the sample. The funnel g is washed
several times with a few drops of sulfuric acid, which are successively introduced into G. The
total liquid introduced should not exceed ten to fifteen cubic centimeters, of which the
greater part should be sulfuric acid. The rubber tube connecting G and F is carefully closed
with a clamp and G violently shaken for a few minutes until no further evolution of nitric oxid
takes place. In shaking, the apparatus should be so held as to prevent the escape of the
mercury from the small tube i by keeping it closed with the finger or drawing over it a rubber
cap.
After the evolution of the gas has ceased, the tube e, Fig. 83, is brought into contact with i,
Fig. 84, and the two are joined by a tight-fitting piece of rubber tubing in such a way as to
exclude any particle of air. The tube F, Fig. 84, is lifted and B and C, Fig. 83, depressed. On
carefully opening the cocks h and b and bringing i and e into union, the gas is passed from G
into A. When all the gas has entered A and the acid mixture from G has reached b the latter is
closed, and also h. The apparatus G is disconnected and removed. The gas in A is then
reduced to normal conditions by manipulating the reduction tube C in the manner already
described.
The gas in A is measured dry by reason of having been generated in presence of rather
strong sulfuric acid. Consequently, for this operation the adjustment of the volume of gas in C
should be made in contact with a drop of strong sulfuric acid. In order to make the readings,
a quantity of material must be taken which will give less than thirty or from 100 to 140 cubic
centimeters of nitric oxid.
 The quantities of the different compounds of nitric acid corresponding
to the number of cubic centimeters of nitric oxid, measured under
standard conditions, are shown in the following table:

Figure 84.

Lunge’s Analytic
Apparatus.

CORRESPONDING TO
———————————— ———————————— ————————————
Cubic Weight in N₂O₃ in milligrams. HNO₃ in milligrams. NaNO₃ in milligrams.
centimeters milligrams.
of NO.
1 1.343 1.701 2.820 3.805
2 2.682 3.402 5.640 7.610
3 4.029 5.103 8.460 11.415
4 5.372 6.804 11.280 15.220
5 6.715 8.506 14.100 19.025
6 8.058 10.206 16.920 22.830
7 9.401 11.907 19.740 26.635
8 10.744 13.608 22.560 30.440
9 12.087 15.309 25.380 34.245

476. Utility of the Method.—Where it is desirable that the nitric oxid method be used,
and at the same time heating be avoided, the decomposition of a nitrate by means of metallic
mercury and sulfuric acid affords a convenient and accurate procedure. But, as a rule, there is
no objection to the application of the lamp, and in such cases the mercury method appears to
have no advantage over the ferrous chlorid process. Nevertheless, in the hands of a skilled
worker the results are reliable, and the process is a quicker one, on the whole, than by
distillation with ferrous chlorid and hydrochloric acid. This method, however, can not be
recommended as in any way superior to the reduction methods to be hereinafter described.
 ESTIMATION OF NITRIC ACID BY OXIDATION OF A
COLORED SOLUTION.
477. Method of Boussingault.—The process for the estimation of nitric acid by the
decoloration of a solution of indigo is due originally to Boussingault.[305] In this method the
extract, obtained by washing slowly 200 grams of soil until the filtrate amounts to 300 cubic
centimeters, is evaporated until its volume is no greater than two or three cubic centimeters,
and it is transferred to a test-tube, with washings, and again evaporated in the tube until the
volume is not greater than that last mentioned. A few drops of solution of indigo are added,
and then two cubic centimeters of pure hydrochloric acid; the whole is then heated. As the
color of the indigo disappears more is added. When the color ceases to fade, the liquid in the
test-tube is concentrated by boiling. If concentration fail to destroy the blue or green color,
another one-half cubic centimeter of hydrochloric acid is introduced. The reaction is
completed when neither concentration nor fresh addition of hydrochloric acid destroys the
excess of indigo present. The color produced by a small excess of indigo is a bright sap-green;
this tint is the final reaction sought. The small excess of indigo necessary to produce a green
color is deducted in every experiment.
When more than mere traces of organic matter are present, Boussingault advises that the
nitric acid be first separated by distillation and then reduced by the indigo solution. For this
purpose the concentrated solution of the nitrate, two or three cubic centimeters, is placed in a
small tubulated retort with two grams of manganese dioxid in fine powder. The retort is next
half filled with fragments of broken glass, over which is poured one cubic centimeter of
concentrated sulfuric acid. The retort is heated carefully by means of a small flame, which is
kept in motion so as to successively come in contact with all parts of the bottom of the retort.
The distillate is received in a graduated test-tube which is kept cool. The distillation is
continued until the vapors of sulfuric acid begin to appear. The apparatus is allowed to cool,
the stopper of the retort removed, two cubic centimeters of water introduced, and the
distillation again made until fumes of sulfuric acid are again seen. The distillation with water
is made twice in order to remove every trace of nitric acid from the retort. The distillate is
neutralized with a solution of potassium hydroxid and concentrated to two cubic centimeters,
and the nitric acid estimated in the manner already described. The manganese dioxid used
should be previously well washed and the sulfuric must be free of nitric acid.
Preparation of the Indigo Solution.—Fifty grams of indigo in fine powder are digested for
twenty-four hours, at 40°, in a liter of distilled water. The water is then poured off and
replaced with a fresh supply. After the second decantation the residue is treated with 750
cubic centimeters of equal parts of water and pure concentrated hydrochloric acid and boiled
for an hour. After cooling, the undissolved portion is collected on a filter and washed at first
with hot, and afterwards with cold water, until the filtrate is no longer colored and is free of
acid. The dried residue is treated with ether under a bell-jar, or in a continuous extraction
apparatus, until the ether is only of a faint blue tint. The fifty grams of indigo at first taken
will give about twenty-five grams of the purified article, which, however, will still leave a little
ash on combustion.
Solution in Sulfuric Acid.—Five grams of the purified indigo are placed in a flask having a
ground-glass stopper, treated with twenty-five grams of fuming sulfuric acid, and allowed to
digest two or three days at a temperature of from 50° to 60°. From seventy to 200 drops of
the solution thus made are placed in 100 cubic centimeters of water for use in the process.
Standardization of the Indigo Solution.—The solution as prepared above is standardized
by a solution of one gram of pure potassium nitrate in 1,000 cubic centimeters of distilled
water. The oxidation of the indigo solution is accomplished as described above. For this
strength of standard nitrate solution two cubic centimeters are taken corresponding to two
milligrams of potassium nitrate. The indigo solution for this strength should have only
twenty drops of the sulfuric acid solution of indigo to 100 cubic centimeters of water. If
twenty grams of potassium nitrate are taken for 1,000 cubic centimeters of the standard
solution then 200 drops of the sulfindigotic acid should be used to 100 cubic centimeters of
water.
478. Method of Marx.—As usually practiced, the indigo method is conducted according
to the variation described by Marx.[306] There are required for the process the following
reagents and apparatus:
a. A solution of pure potassium nitrate containing 1.8724 grams per liter. One cubic
centimeter of the solution is equivalent to one milligram of nitric anhydrid (N₂O₅).
b. A solution of the best indigo carmine in water which should be approximately
standardized by solution in the manner described hereafter, and then diluted so that six to
eight cubic centimeters equal one milligram of nitric acid.
c. Chemically pure sulfuric acid of specific gravity 1.842, perfectly free from sulfurous and
arsenious acids and nitrogen oxids.
d. Several thin flasks of about 200 cubic centimeters capacity.
e. A small cylindrical measure holding fifty cubic centimeters and divided into cubic
centimeters.
f. A Mohr’s burette divided into tenths of a cubic centimeter.
g. A twenty-five cubic centimeter pipette or another burette.
h. A five cubic centimeter pipette divided into cubic centimeters or half cubic centimeters.
i. A measuring flask of 250 cubic centimeters capacity.
Preliminary Trial.—Twenty-five cubic centimeters of the sample are transferred to a flask;
the fifty cubic centimeter measure is filled with sulfuric acid and the burette with indigo
solution. The sulfuric acid is added to the sample all at once, shaken for a moment, and the
indigo run in as quickly as possible with shaking until a permanent greenish tint is produced.
If the sample do not require more than twenty cubic centimeters of indigo solution of the
above strength, it can be titrated directly, otherwise it must be diluted with a proper quantity
of pure water, and subjected again to the preliminary trial.
The Actual Titration.—(1) Twenty-five cubic centimeters of the sample properly diluted if
necessary, are measured and poured into a flask, and as much indigo as was used in the
preliminary trial, is added; a quantity of sulfuric acid, equal in volume to the liquid in the
flask, is added all at once, the mixture shaken, and indigo solution run in quickly out of the
burette until the liquid remains permanently of a greenish tint.
(2) The last experiment is repeated as often as may be necessary adding to the water at first
half a cubic centimeter less indigo than the total quantity used previously, afterwards
proceeding as in (1) until the final test shows too little indigo used.
(3) From the rough titration of the indigo, calculate the amount of potassium nitrate
solution corresponding with the indigo solution used in (2), multiply the result by ten,
transfer this quantity of the standard nitrate solution to a 250 cubic centimeter flask, fill with
pure water to the mark, and titrate twenty-five cubic centimeters of this fluid with indigo as
in (2). If the quantity of indigo solution used is nearly the same as that required in (2), its
exact value may be calculated, but if it is not, another nitrate solution may be made up in the
250 cubic centimeter flask, more closely resembling the sample in strength, and the titration
with the indigo solution must be repeated.
(4) If the water contain any considerable amount of organic matter, it must first be
destroyed by potassium permanganate. In this case, the estimation of the organic matter and
nitric acid may be conveniently combined.
The use of permanganate in the above case is likely to introduce an error as has been
shown by Warington. The method therefore can not be recommended in the presence of
organic matter.
479. Method of Warington.—The modification of the indigo method as used by
Warington, applicable only in absence of organic matter, is the one chiefly employed in
England.[307]
Instead of the ordinary indigo of commerce, indigotin is used. The normal solution of the
coloring matter is made of such a strength as to be equivalent to a solution of potassium
nitrate containing 0.14 gram of nitrogen per liter. Where large quantities of the coloring
matter are to be used it is advisable to prepare it about four times the strength given above
and then dilute it as required. Four grams of sublimed indigotin will furnish more than two
liters of the color solution.
The solution is prepared as follows:
Four grams of indigotin are digested for a few hours with five times that weight of
Nordhausen sulfuric acid, diluted with water, filtered, and made up to a volume of two liters.
The strength of the indigotin solution is determined with a solution of potassium nitrate of
the strength mentioned above. The process is performed as follows:
From ten to twenty cubic centimeters of the standard nitrate solution are placed in a wide-
mouthed flask of about 150 cubic centimeters capacity. A portion of the indigotin solution is
next added, such as will be deemed sufficient for the process, and the whole is well mixed.
Strong sulfuric acid is next measured out from a burette into a test-tube, in volume equal to
the united volumes of the nitrate solution and indigotin. The whole of the sulfuric acid is then
poured as quickly as possible, into the solution in the flask and rapidly mixed, and the flask
transferred to a calcium chlorid bath, the temperature of which should be maintained at 140°.
It is essential to the success of the operation that the sulfuric acid should be mixed with the
greatest rapidity. It should be poured in at once and the whole well shaken without waiting
for the test-tube containing the acid, to drain. The flask should be covered by a watch-glass
while it is held in the bath. As soon as the larger part of the indigotin is oxidized the flask in
the bath should be gently rotated. With very weak solutions of nitrate it may be necessary
sometimes to keep the flask in the bath for five minutes. When the indigo color is quickly
discharged it shows the presence of nitric acid in considerable excess and a considerably
larger quantity of indigo must be taken in the next experiment. The experiments are
continued until just the quantity of indigo necessary to consume the nitric acid is taken, the
amount of indigo being in very slight excess, not exceeding one-tenth cubic centimeter of the
indigo solution used. The tint produced by the small excess of indigo remaining is best seen
by filling the flask with water. On substances of approximately known strength about four
experiments are usually necessary to determine the amount of indigo to be taken, but with
unknown substances a larger number may be necessary.
Usually in determinations of this kind it is directed to use double the volume of sulfuric
acid mentioned above. In this case not only is the quantity of indigo oxidized much greater
than with a smaller portion of acid, but the prejudicial effect of organic matter is also greater
than when the smaller quantity of acid is employed.
An indigo solution standardized as above is strictly to be used for a solution of nitrate of
the strength employed during the standardization. The quantity of indigo oxidized in
proportion to the nitric acid present diminishes as the nitrate solution becomes more dilute.
Instead of determining this during each series of experiments it may be estimated once for all
and a table of corrections used.
The following table is based upon experimental determinations:
 Strength of Indigo Difference Nitrogen Difference Difference in the
niter solution required, between corresponding between the nitrogen values for a
used. cubic amounts of to one cubic nitrogen difference of one cubic
centimeters. indigo, cubic centimeter of values, gram. centimeter in the
centimeters. indigo, gram. amount of indigo, gram.
⁸⁄₆₄ Normal 10.00 0.000035000
⁷⁄₆₄ „ 8.71 1.29 0.000035161 0.000000161 0.000000125
⁶⁄₆₄ „ 7.43 1.28 0.000035330 0.000000169 0.000000132
⁵⁄₆₄ „ 6.14 1.29 0.000035627 0.000000298 0.000000231
⁴⁄₆₄ „ 4.86 1.28 0.000036008 0.000000381 0.000000298
³⁄₆₄ „ 3.57 1.29 0.000036763 0.000000756 0.000000586
²⁄₆₄ „ 2.29 1.28 0.000038209 0.000001445 0.000001129
¹⁄₆₄ „ 1.00 1.29 0.000043750 0.000005541 0.000004295

The table is used as follows:

Suppose that twenty cubic centimeters of water under examination have required 5.36
cubic centimeters of indigo solution for the oxidation of the nitric acid contained therein. By
inspection of the table it is seen that this number is five-tenths cubic centimeter above the
nearest quantity given; viz., 4.86 cubic centimeters. From the last column in the table it is
found that the correction for five-tenths cubic centimeter of indigo solution is 0.000000149
cubic centimeter, being half that for the one cubic centimeter given in the table. This is to be
subtracted from the unit value in nitrogen given in the first “gram” column of the table; viz.,
0.000036008. It is thus seen that the 5.86 cubic centimeters of indigo solution are
equivalent to 0.000035859 gram of nitrogen per cubic centimeter. The water under
examination, therefore, contains nine and six-tenths parts of nitrogen as nitric acid per
million.
Attention must also be paid in standardizing indigo solutions to the initial temperature of
the solutions. A rise in the initial temperature will be attended by a diminution in the
quantity of indigo oxidized. Experiments with a room temperature of 10° and a room
temperature of 20°, being the initial temperatures of the experiments, showed that at the
higher temperature the amount of indigo consumed was about five per cent less when the
strong solutions of nitrate were employed. The indigo solution should, therefore, be
standardized at the same temperature at which the determinations are made.
If twenty cubic centimeters of the standard nitrate solution employed be used in setting the
indigo solution, this standard will enable the operator to determine nitric acid up to 17.5
parts of nitrogen per million in water or soil extracts.
The presence of an abundance of chlorids in the water under examination tends to
diminish the content of nitric acid found, and also tends to introduce an error, which is
sometimes of a plus and sometimes of a minus quantity, according to the strength of the
nitric acid present. The reaction is shortened in weak solutions by the presence of chlorids,
and the quantity of indigo consumed is consequently increased. The error introduced by
chlorids is usually of an insignificant nature.
On account of the interference of organic matters with the reaction of indigo it is not of
much use in the examination of nitrates washed out of soils, although in some cases the
results may be quite accurate. This method must, therefore, be considered as applicable, in
general, to waters or soil extracts which contain little or no organic matter.
In analytical work pertaining particularly to agriculture, the use of the indigo method for
determining nitric acid has been largely employed, both in the analyses of soil extracts and
drainage and irrigation waters. The method, however, can hardly survive as an important one
in such work in competition with more modern and speedy processes of analysis.
 DETERMINATION OF NITRIC NITROGEN BY REDUCTION
TO AMMONIA.
480. Classification of Methods.—When nitrogen is present in a highly oxidized state,
e. g., as nitric acid, it may be quickly and accurately estimated by reduction to ammonia. This
action is effected by the reducing power of nascent hydrogen, and this substance may be
secured in the active state by the action of an acid or alkali on a metal, or by means of an
electric current. The processes depending on the use of a finely divided metal in the presence
of an acid or alkali have come into general use within a few years, and are now employed
generally instead of the more elaborate estimations depending on the use of copper oxid or
indigo.
The typical reaction which takes place in all cases is represented by the following equation:
2HNO₃ + 8H₂ = 2NH₃ + 6H₂O.
The method will be considered under three heads; viz., 1. Reduction in an alkaline solution;
2. Reduction in an acid solution; 3. Reduction by means of an electric current.
In the first class of processes the reduction and distillation may go on together. In the
second class the reduction is accomplished first and the distillation effected afterwards, with
the addition of an alkali. In the third class of operations the reduction is accomplished by
means of an electric current and the ammonia subsequently obtained by distillation, or
determined by nesslerizing. These processes may be applied to rain and drainage waters, and
to soil extracts. On account of the ease with which the analyses are accomplished, the short
time required and the accuracy of the results, the reduction methods for nitrates have already
commended themselves to analysts, and are quite likely to supersede all others for practical
use where weighable quantities of nitrates are present. For the minute traces of nitrates
found in rain and drainage waters, and in some soil extracts, the reduction method may also
be applied, but in these cases the ammonia which is formed must be determined by
colorimetry (nesslerizing) and not by distillation. The processes about to be described are
especially applicable to the examination of soils and waters rich in nitrates.
 REDUCTION IN ALKALINE SOLUTIONS.
481. Provisional Method of the Association of Official Agricultural Chemists.
[308]
—
Extraction of the Nitrates.—Place one kilogram of the dried soil, calculated to water-free
substance, on a percolator of glass or tin. Moisten the soil thoroughly with pure distilled
water, and allow to stand for half an hour. Add fresh portions of pure distilled water until the
filtrate secured amounts to one liter. If the first filtrate be cloudy before use it may be
refiltered.
Qualitative Test for Nitrates.—Evaporate five cubic centimeters of the soil extract in a
porcelain crucible, having first dissolved a small quantity of pure brucin sulfate therein.
When dry, add to the residue a drop of concentrated sulfuric acid free of nitrates. If the
nitrate calculated as potassium nitrate does not exceed the two-thousandth part of a
milligram only a pink color will be developed; with the three-thousandth part of a milligram a
pink color with reddish lines; with the four-thousandth part of a milligram a reddish color;
with the five-thousandth part of a milligram a distinct red color.
Estimation of the Nitrates.—Evaporate 100 cubic centimeters of the soil extract to dryness
on a steam-bath. Dissolve the soluble portions of the residue in 100 cubic centimeters of
ammonia-free distilled water, filtering out any insoluble residue. Place the solution in a flask,
add ten cubic centimeters of sodium amalgam, stopper the flask with a valve which will
permit the escape of hydrogen, and allow to stand in a cool room for twenty-four hours. Add
fifty cubic centimeters of milk of lime and titrate the ammonia produced by distillation, with
standard acid and estimate as nitrogen pentoxid. Where the amount of ammonia is small,
nesslerizing may be substituted for titration.
Preparation of Sodium Amalgam.—Place 100 cubic centimeters of mercury in a flask of
half a liter capacity; warm until paraffin will remain melted over the surface; drop
successively in the paraffin-covered mercury, pieces of metallic sodium of the size of a pea
until 6.75 grams have united with the mercury. The amalgam contains then 0.5 per cent of
metallic sodium and may be preserved indefinitely under the covering of paraffin.
482. Method of the Experiment Station at Möckern.[309]—The principle of this
reaction is based on the reducing action exercised by nascent hydrogen on a nitrate, the
hydrogen being generated by the action of soda-lye on a mixture of zinc dust and finely
divided iron.
Ten grams of nitrate are dissolved in 500 cubic centimeters of water. Of this solution
twenty-five cubic centimeters, corresponding to one-half gram, are placed in a distillation
flask of about 400 cubic centimeters capacity, 120 cubic centimeters of water added, and
about five grams of well-washed and dried zinc dust and an equal weight of reduced iron. To
the solution are added eighty cubic centimeters of soda-lye of 32° B. The flask is then
connected with the condensing apparatus and the distillation carried on synchronously with
the reduction, the ammonia being collected in twenty cubic centimeters of titrated sulfuric
acid. The distillation is continued from one to two hours, or until 100 cubic centimeters have
been distilled, and the remaining sulfuric acid is titrated in the usual way. Soil extracts and
sewage waters should be concentrated until they have approximately the proportion of
nitrates given above.
483. Method of Devarda.—The inconvenience due to slow action and other causes,
arising from the use of pure metals in the reduction of nitrates to ammonia, has been
overcome, to some extent, by Devarda, by use of an alloy, in a state of fine powder, consisting
of aluminum, copper, and zinc.[310] The alloy consists of forty-five per cent of aluminum, fifty
per cent of copper, and five per cent of zinc. In dissolving, the copper is left in a finely divided
state, which is a great help in distillation in preventing bumping.
The analytical process is carried out as follows: The solution containing the nitrate, in
quantity equivalent to about one-half gram of potassium nitrate, is placed in a flask having a
capacity of about one liter, and diluted with sixty cubic centimeters of water and five cubic
centimeters of alcohol, and then forty cubic centimeters of caustic potash solution added of
specific gravity one and three-tenths. From two to two and one-half grams of the alloy,
described above, are introduced, and the flask attached to a condenser with a receiver
containing standard acid. The connection between the flask and the condenser is made by
means of a tube having on the limb next the flask a bulb filled with glass beads to prevent the
contents of the flask splashing over into the receiver, and on the other limb another bulb to
prevent the acid in the receiver finding its way into the distillation flask, should regurgitation
occur. When the flask has been thus connected with the condenser it is gently heated for half
an hour, at the end of which time the evolution of hydrogen will have slackened or ceased,
and then the distillation is begun, at first cautiously, until the zinc of the alloy has completely
dissolved, and then more vigorously, the time necessary being about twenty minutes from the
time when the contents of the flask begin to boil. The distillate is caught in standard acid and
the ammonia determined by backward titration in the ordinary way. It is to be noted that the
strength of the alkali used is of importance, as if it be too strong, the action on the alloy is
unduly vigorous at the beginning of the operation, and if too weak, the contents of the flask
have to be heated overmuch, the result in both cases being the formation of a fine spray of
caustic solution, which is very difficult to stop, even with complicated washing attachments to
the distilling flask. The test analyses on pure nitrates are satisfactory. This method has been
used with satisfaction in the laboratory of the Department of Agriculture, but does not appear
to have any special advantage over the process of Ulsch, to be described further on.
484. Variation of Stoklassa.—Stoklassa has subjected the method of Devarda to a
comparative test with the following methods:[311]
1. Wagner’s Schloesing-Grandeau method.
2. Lunge’s nitrometer method.
3. Stutzer’s method.

Figure 85. Stoklassa’s Nitric Acid Apparatus.

 The reduction takes place in a copper erlenmeyer, A, Fig. 85, in which, in addition to the
solution containing the nitrate, are placed 200 cubic centimeters of water, forty cubic
centimeters of potassium hydroxid solution of 33° B., five cubic centimeters of alcohol, and
finally two and one-half grams of the finely powdered Devarda alloy. The distillate passes
through a tube, B, filled with glass pearls and into the condenser D, through the bulbs, C C′.
After the flask is connected with the distilling apparatus, it is gently warmed and the
reduction is ended in about twenty minutes. The ammonia which is formed is then distilled
into E, containing the standard acid, S, requiring about twenty minutes more. The
comparative results given, show that the Devarda method is equally as accurate as any of the
other methods mentioned, giving practically theoretical results.
In so far, however, as speed of an analysis, is concerned the first place is awarded to the
Lunge nitrometer method, with which a complete analysis can be made in from thirty to forty
minutes. In the second rank, so far as speed is concerned, the Devarda method is
recommended. All the methods give accurate results.
485. Method of Sievert.[312]—Two grams of potassium or sodium nitrate are dissolved
and made up to 1,000 cubic centimeters. Fifty cubic centimeters of the solution are placed in
a 600 cubic centimeter flask and diluted with fifty cubic centimeters of water, and from
eighteen to twenty grams of caustic alkali added. After the alkali is dissolved, seventy-five
cubic centimeters of ninety-six per cent alcohol are added and a few pieces of bone-black to
prevent foaming. From ten to fifteen grams of zinc or iron dust are then added to the flask
which is closed and connected with a ᥩ tube holding about 200 cubic centimeters, which
contains about ten cubic centimeters of normal sulfuric acid. This ᥩ tube is kept cool by
being immersed in water. The whole mixture is now allowed to stand for three or four hours
and then the alcohol is distilled slowly and the ammonia formed by the reduction of the
nitrates is carried over with it. The distillation lasts for about two hours. The contents of the
ᥩ tube are carefully rinsed into a dish and the excess of sulfuric acid titrated with one-fourth
normal soda-lye.
For soil extracts and substances containing unknown quantities of nitric acid, a
preliminary test will indicate approximately the amount thereof, and this will be an
indication for the quantity to be used in the analysis.
The method of Stutzer differs from the foregoing in the substitution of aluminum dust
instead of iron or zinc.[313] The reducing power of aluminum, however, varies greatly
according to the method in which the metal has been prepared. Pure aluminum prepared by
the electric method, reduces the nitric acid much less vigorously than the metal prepared by
the older methods of fusion with sodium. For this reason the method of Stutzer is not to be
preferred to that of Sievert.
 REDUCTION IN AN ACID SOLUTION.
486. Variation of the Sodium Amalgam Process.—This method is described by
Monnier and Auriol.[314]
The principle of the operation depends on the reduction of
the dissolved nitrate by titrated sodium amalgam in
presence of an acid, and the estimation of the quantity of
nitric acid present from the deficit in the volume of
hydrogen. The apparatus employed is conveniently mounted
as shown in Fig. 86. The brass vessel A is movable by means
of the cord on the pulley B, in such a way as to be fixed at
any required altitude. It is filled with water and connected
by a rubber tube to the cooling tube D. Within the cooling
tube there is a graduated cylinder open at its lower end. Its
upper end is connected directly with the apparatus C. The
cooling tube D has a small side tube, c, near its upper end,
by means of which the air can enter or escape when the
position of A is changed. The apparatus C, in which the
reaction takes place, is a glass cylinder. Its upper end is
continuous with the ⟙ tube provided with the stop-cocks a
and b. One arm of the ⟙ permits connection with the
graduated measuring tube by means of a rubber union. The
lower end of C is closed with a large hollow ground-glass
stopper, carrying a small receptacle within, so that it forms
two separate water-tight compartments, open at the top.
The sodium amalgam is prepared as follows:
In a clay crucible are heated 400 grams of mercury, and,
little by little, with constant stirring, four grams of dry
Figure 86. Variation of the sodium are added. When cold, the amalgam is placed in a
Sodium Amalgam Process.
burette, having a ground-glass stopper, and covered with
petroleum. The strength of the amalgam is established in the
following manner. A small glass thimble, ground even at the top, is filled with the amalgam
and struck off even with a ground-glass straight edge. In this way the same quantity of
amalgam is taken for each test. This measured portion of the amalgam is placed in the inner
vessel of the glass stopper to C. Ten cubic centimeters of water, containing sixty centigrams of
tartaric acid, are placed on the outer ring of the glass stopper, which is then inserted, well
oiled, in C, closing it air- and water-tight. The tartaric acid solution also carries a piece of
litmus paper, so that its constant acidity may be insured. The vessel A is then fixed in a
position which brings the water in the graduated burette and tube D exactly to the 0 mark.
The cock a is next closed, b opened, and C is inverted until all the amalgam is poured into the
solution of tartaric acid. The evolved hydrogen mixed with the air contained in the apparatus,
is passed into the graduated burette. After fifteen minutes, the reaction is ended. The water
level within and without the graduated tube is restored and the volume of gas evolved noted
and reduced by the usual tables to 0° and 760 millimeters pressure of the barometer.
An amalgam prepared as above will give about three cubic centimeters of hydrogen for
each gram. The thimble should hold from twelve to fifteen grams.
The estimation of nitric acid should be made in a solution containing about one-tenth per
cent of nitrate. Ten cubic centimeters are taken, to which six-tenths gram of tartaric acid is
added, and placed in the outer part of the glass stopper. The rest of the process is conducted
exactly as described above. The deficit in hydrogen is calculated to nitrogen pentoxid.
The reduction by sodium amalgam is not so convenient a form of estimating nitric acid as
many of the other forms of using nascent hydrogen. As practiced by calculating from the
deficit of hydrogen, however, it has some advantages by reason of the fact that no heating is
required. The presence of organic neutral bodies, or even those of an acid nature, like humus,
does not, therefore, interfere with the work. Likewise, mineral bodies in solution, which are
not reduced by nascent hydrogen, do not interfere with the accuracy of the reaction.
487. Method of Schmitt.—In the method of Schmitt forty cubic centimeters of glacial
acetic acid are placed in a flask of 600 cubic centimeters content, and fifteen grams of a
mixture of zinc and iron dust added.[315] To this a quantity of the solution containing the
nitrate, representing about half a gram of the pure nitrate, is added with constant shaking, in
portions which do not evolve hydrogen too rapidly. After about fifteen minutes when the
evolution of nitrogen has somewhat diminished, an additional fifteen grams of the metal dust
are added. If the contents of the flask should become thick they can be diluted with thirty
cubic centimeters of water. The reduction is complete in from thirty to forty minutes. The
contents of the flask are now saturated with enough soda-lye not only to neutralize the excess
of acetic acid, but to keep the zinc hydroxid also in solution. For this purpose about 200 cubic
centimeters of soda-lye of 1.25 specific gravity are necessary. The ammonia is obtained by
distillation into standard acid in the usual way.
488. Method of Ulsch.—In practice the method of Ulsch has come into general use.[316]
For the determination of nitrogen by this method half a gram of saltpeter or four-tenths
gram of sodium nitrate is taken and dissolved in twenty five cubic centimeters of water, in a
flask with a content of about 600 cubic centimeters. Five grams of iron reduced by hydrogen,
and ten cubic centimeters of sulfuric acid diluted with two volumes of water are then added
to the flask. To avoid mechanical losses during the evolution of hydrogen a pear-shaped glass
stopper is hung in the neck of the flask. After the first violent evolution of hydrogen has
passed, the flask is slowly heated until in about four minutes it is brought to a gentle boil. The
boiling is continued for about six minutes when the reduction is complete. About fifty cubic
centimeters of water are then added; also an excess of soda-lye and a few particles of zinc and
the ammonia is distilled and collected in standard acid in the usual way.
The method of Ulsch can also be applied, according to Fricke, to the analysis of nitrates
contained in drinking and drainage waters, and it is regarded by him as one of the best
methods to be employed in such investigations.[317]
The method of Ulsch in this laboratory has given entirely satisfactory results, and is
generally used in preference to other methods in cases where a considerable quantity of
nitrates is present. It is based on the following reactions:

2KNO₃ + H₂SO₄ = K₂SO₄ + 2HNO₃

2HNO₃ + 8H₂ = 2NH₃ + 6H₂O
2NH₃ + H₂SO₄ = (NH₄)₂SO₄.