BIOLOGICAL SOIL CRUST CARPET

ENGINEERING

SEMINAR REPORT

Submitted by

DEVIKA P
SCT19BT022

To
Abdul Kalam Technological University
in partial fulfillment for the award of the degree of

Bachelor of Technology
in
Biotechnology and Biochemical Engineering

Department of Biotechnology and Biochemical Engineering,

SREE CHITRA THIRUNAL COLLEGE OF ENGINEERING
DECEMBER - 2022
 DECLARATION

I undersigned hereby declare that the seminar report “BIOLOGICAL SOIL CRUST
CARPET ENGINEERING”, submitted for partial fulfillment of the requirements for
the award of degree of Bachelor of Technology of the APJ Abdul Kalam Technological
University, Kerala is a bonafide work done by me under supervision of Mrs.Ani Lawrence.
This submission represents my ideas in my own words and where ideas or words of others
have been included; I have adequately and accurately cited and referenced the original
sources. I also declare that I have adhered to ethics of academic honesty and integrity
and have not misrepresented or fabricated any data or idea or fact or source in my
submission. I understand that any violation of the above will be a cause for disciplinary
action by the institute and/or the University and can also evoke penal action from the
sources which have thus not been properly cited or from whom proper permission has
not been obtained. This report has not been previously formed the basis for the award
of any degree, diploma or similar title of any other University.

Place: Thiruvananthapuram
Date: 11/12/2022 DEVIKA P
 DEPARTMENT OF BIOTECHNOLOGY AND
BIOCHEMICAL ENGINEERING

SREE CHITRA THIRUNAL COLLEGE OF ENGINEERING,

THIRUVANANTHAPURAM-695018.

CERTIFICATE
This is to certify that the report entitled BIOLOGICAL SOIL CRUST CAR-
PET ENGINEERING submitted by DEVIKA P (SCT19BT022), to the APJ Abdul
Kalam Technological University in partial fulfillment of the B.Tech. degree in Biotech-
nology and Biochemical Engineerung is a bonafide record of the seminar work carried
out by her under our guidance and supervision. This report in any form has not been
submitted to any other University or Institute for any purpose.

SEMINAR COORDINATOR SEMINAR GUIDE

Smt.Susmithakumari S Smt.Ani Lawrence
Assistant professor Assistant professor
Department of Biotechnology Department of Biotechnology
and Biochemical Engineering and Biochemical Engineering
HEAD OF DEPARTMENT
Prof.Dr.K.B Radhakrishnan
professor
Department of Biotechnology
and Biochemical Engineering
 ACKNOWLEDGEMENT

I take this opportunity to write a few words and thank some people who have been of
great help and source of encouragement.

I am thankful to our Prof.Dr.V.Syam Prakash (Principal; Sree Chitra Thirunal Col-

lege of Engineering) for rendering great encouragement and providing this opportunity.

I am also obliged to Prof.Dr.K. B Radhakrishnan, Professor and Head of the De-

partment (Department of Biotechnology and Biochemical engineering), SCT College of
engineering, for the support and encouragement provided by him to make this seminar
a success.

I am deeply indebted to my Guide, Mrs.Ani Lawrence, Assistant professor, Depart-

ment of Biotechnology and Biochemical engineering, SCT College of engineering, whose
help, stimulating suggestions and encouragement helped me throughout the duration of
the study.

I would like to express my gratitude to all those who helped me to complete this seminar.
I would like to thank the faculty members of the Department of Biotechnology and
Biochemical engineering, SCT College of engineering, Thiruvananthapuram, for giving
me the opportunity and facilities to undertake the study of this topic and for its successful
completion.

DEVIKA P
 Contents

List of Figures ii

List of Tables iii

1 INTRODUCTION 1

2 BIOLOGICAL SOIL CRUST COMPONENTS 2

3 TYPES OF BIOLOGICAL SOIL CRUST IN THE FIELD 4

4 DEVELOPMENT OF CYANOBACTERIAL BIOCRUST CARPET 6

4.1 Strain Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4.2 Strain Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4.3 Inoculant Production And Dispersion . . . . . . . . . . . . . . . . . . . . 7

4.4 Biocrust Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

5 ROLE OF EXOPOLYSACCHARIDES (EPS) MATRIX IN THE BIOCRUST

COMMUNITY 10

6 EVALUATION OF THE INOCULATION EFFECT 12

7 FACTORS AFFECTING INOCULATION 13

7.1 Soil salinity and pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

7.2 Substrate Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . 13

7.3 Water Availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

7.4 Inoculum Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

7.5 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
 7.6 Light Intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

8 IMPORTANCE OF BIOLOGICAL CRUSTS 16

9 CONCLUSION 17

REFERENCES 18

APPENDIX 19

i
 List of Figures

1.1 Biocrust formed in the Colorado Plateau, Utah . . . . . . . . . . . . . . 1

4.1 Biocrust Formation : Fig(A) Upper view of sand microcosms inoculated

with the gliding strain L.ohadii, and Fig(B) with the non-gliding strain
Schizothrix delicatissima AMPL0116 . . . . . . . . . . . . . . . . . . . . 9

5.1 EPS-sheath collating close sand particles . . . . . . . . . . . . . . . . . . 10

ii
 List of Tables

6.1 Set of analytical methods to assess inoculant establishment and growth in

the substrate, and the stability of induced biocrusts . . . . . . . . . . . . 12

iii
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Chapter 1
INTRODUCTION

Biological soil crust are complex communities of living organisms such as

cyanobacteria, green algae, lichens, mosses, microfungi and other bacteria. These crusts
are also called cryptogamic, cyptobiotic ,microbiotic or microphytic soil crusts.This thin
layer of living material is formed in the uppermost millimetres of soil where soil particles
are aggregated by a community of those highly specialized organisms.

Biological soil crust are found primarily in open spaces in the dry and ex-
tremely cold regions of all continents, where harsh conditions inhibit vascular plant pro-
duction. In many areas ,the crust are extra ordinarily well developed and can represent
more than 70 percent of living ground cover.These crusts are key for soil stabilization,
water retention and soil fertility and are recognised as having a major influence on global
ecosystem.

Figure 1.1: Biocrust formed in the Colorado Plateau, Utah

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Chapter 2
BIOLOGICAL SOIL CRUST
COMPONENTS

Biological soil crusts have both macro and microscopic components. Compo-
nents that comprise these crusts are common in desert soils throughout the world.The
components are:

(i) Microfungi

It occur either as free living organism or in mycorrhizal association with plant

roots.Free living microfungi function as decomposers.Fungal filaments (hyphae) bind soil
particles together,increasing soil water-holding capacity.

(ii) Bryophtes

Bryophytes are tiny non-vascular plants.This group includes both mosses

and liverworts.Mosses are usually classified as short annual mosses or tall perennial
mosses.Liverworts can be flat and ribbon- like or leafy.They can reproduce by spore
formation or by asexual fragmentation,and photosynthesize to fix carbon from the atmo-
sphere.

(iii) Bacteria

Bacteria are a diverse group of primitive,single-celled organisms.Bacteria can

be either autotrophic or heterotrophic.Some bacteria contribute to soil fertility by fixing
nitrogen.Others are important in decomposition.

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(iv) Green-algae

They are light green to black, single-celled, photosynthetic organisms.Algae

are difficult to observe without a microscope but sometimes give the soil surface a green
tint.Their growth period is often linked to cool,moist weather,and they may be difficult
to detect when dry.

(v) Lichens

Lichens are fungi that capture and cultivate algae or cyanobacteria.They il-
lustrate the concept of symbiosis.Lichens occur in a variety of colors, including green,
red, brown, white, and black.

(vi) Cyanobacteria/Blue green algae

They are primitive filamentous or single-celled bacteria that can photosynthe-

size and, under anaerobic conditions, fix atmospheric nitrogen.They can be heterocystic
or non-heterocystic.

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Chapter 3
TYPES OF BIOLOGICAL SOIL CRUST
IN THE FIELD

Biological soil crusts are typically made up of a variety of species and mor-
phological groupings, unless the crust is at an early stage of succession. The crust will
typically be dominated by one or two morphological groupings.Typical morphological
groupings include the following:

(1) Cyanobacterial Crusts

Cyanobacterial crusts are dark colored or black.The organisms may be seen

as black filaments on and near the soil surface when the soil is damp.

(2) Greenalgal Crusts

Although they are not always visible, green algal crusts can sometimes be
seen as a green tint on the surface of moist soil.

(3) Moss Crusts

Moss crusts can be seen as a fuzzy carpet with flecks of green, gold, brown,
or even black.

(4) Liverworts

Liverworts are difficult to find and typically co-exist in a mosaic with other
dominant organisms. Tiny black ribbons can be seen under a hand lens under close
inspection; they become more noticeable when moistened.

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(5) Lichen Crusts

Lichen crusts can be recognised by their wide variety of forms and hues.
An anatomical gradient between low, straightforward morphologies and taller, three-
dimensional growth forms is formed by the lichen morphological groupings.

• Crustose lichens are flat and fused to the substrate.

• Gelatinous lichens are usually black and may appear flat or three-dimensional.

• Squamulose lichens occur as small individual flakes or scales that often grow in
colonies or clusters.

• Foliose lichens are leaf-like and loosely appressed to the substrate.

• Fruticose lichens are three dimensional and are often upright, branched, or thread-
like.

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Chapter 4
DEVELOPMENT OF
CYANOBACTERIAL BIOCRUST
CARPET

Cyanobacteria play a crucial role in the stability and defence against erosive
forces that biocrusts provide. They are also in charge of carbon and nitrogen fixation,
which enriches soils with macro- and micronutrients. Because of these cyanobacteria’s
contributions to the ecosystem, it is crucial to comprehend their global makeup and role in
nature.[4] The selection of cyanobacterial strains to produce the inoculum must be based
on the soil type and environmental conditions of the site to be reclaimed to properly
tailor the treatment. The general procedural step for the development of cyanobacterial
biocrust carpet includes:

4.1 Strain Isolation

Next generation 16s rRNA gene sequencing is frequently carried out to deter-
mine the important cyanobacterial species primarily responsible for the local soil coloni-
sation and soil stabilization. This will direct the field sample isolation procedure and
identify the most promising genera and species.Procedure for isolation and purification
involves;

• Field Collection : Collection of soil or biocrust samples with a visible presence of a

photosynthetic activity.

• Enrichment Culture : Sample fragments are suspended in nutrient media until a

diffuse greenish coloration, sign of a cumulative growth,is observed in the medium.

• Isolation Of Cyanobacterial Strain : To obtain monocultures, the green suspension

is repeatedly streaked over agarized nutrient medium.

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• Separation Of Cyanobacterial Isolates : Growing cyanobacterial filaments and

clumps on agar medium can be aseptically collected under a light microscope using
sterilized tweezers.

• Purification Of Cyanobacterial Cultures : To purify the cultures, antibiotic treat-

ments can be carried out to remove eukaryotic and prokaryotic contaminants, fol-
lowed by Imipenem to remove heterotrophic bacteria.

4.2 Strain Selection

The screening of new cyanobacterial candidates is based on the premise that

while some strains have a potent ability to increase soil structural stability, others have
a propensity for biofertilizing properties; typically, most strains only exhibit one of the
two properties, and in very rare cases, neither. It would be desired to develop treatments
for erosion control in the field by identifying the isolates with a remarkable capacity
to stabilise the microcosm surface and enhance the physicochemical conditions of the
substrate in laboratory.

More than one species can be used in the final inoculant to integrate the
various features of various strains. In order to achieve both soil stabilisation and biofer-
tilization, strains with a predominate sediment aggregation capacity can be combined
with a single strain that has a clear biofertilization potential. For instance, scientists
from the Chinese Academy of Sciences have successfully tested the two-strain inoculant
M. vaginatus/S. javanicum (in a 10:1 ratio) to stop desertification and sand dunes.

4.3 Inoculant Production And Dispersion

Methodological and analytical procedures to produce cyanobacterial biomass,the

inoculant [2] and its dispersion involves:

• Biomass Production : Generally obtained by cultivation in liquid medium, increas-

ing volumes stepwise from 50 to 250 mL in Erlenmeyer flasks or in bubble column
photobioreactors.

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• Optimization of the mass-cultivation methodology : Clump formation can be coun-

tered by repeated trichome fragmentation during biomass growth under sterile con-
ditions,or by homogenization.

• Biomass Harvesting : The growth curve of the cyanobacteria strains can be assessed
by quantifying the increment of culture dry weight or chlorophyll a on a time
scale.The concentration of EPS in liquid culture can be assessed by applying the
phenol–sulfuric acid assay (Dubois test).

• Inoculant Production : Eventual association of the biomass to a carrier or an adju-

vant substance to increase the fitness of the cyanobacterial strain and its tolerance
to abiotic stress, increase stickiness or provide nutrient supply.

• Application Of A Hardening Protocol : Application of treatments aiming at in-

creasing the capability of the inoculant to better cope with environmental stresses
when dispersed in the soil.

• Elaboration Of An Optimal Inoculant Dispersal Methodology : Use of a method-

ology to obtain a distribution of the inoculant as uniform as possible to ease the
formation of biocrusts.

4.4 Biocrust Formation

Biocrust formation is revealed by greenish coloration at the soil surface, the

possible presence of green clumps, and the acquisition of two possible surface morpho-
logical patterns. The first morphological pattern is typical of motile strains and is char-
acterized by a smooth topsoil appearance and pronounced thickness development, as in
the case of L. ohadii (Fig A).

When inoculating strains with the ability to migrate into the substrate,
biocrust formation is characterized by a progressive and visible downward migration
of cyanobacterial biomass into the substrate. In contrast, upward filament migration is
observed when the microcosm surface is moistened.[5]Due to the downward migration,
L. ohadii could also be detected in the lower layers of the substrate lower layers of the
substrate.

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The second morphological pattern of biocrust formation, typical of non-motile

strains, is characterized by bio-sedimentary structures (cracks and curls) at the top of
the soil (Fig B), although these features also depend on soil characteristics and abiotic
parameters.

Figure 4.1: Biocrust Formation : Fig(A) Upper view of sand microcosms inoculated with
the gliding strain L.ohadii, and Fig(B) with the non-gliding strain Schizothrix delicatis-
sima AMPL0116

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Chapter 5
ROLE OF EXOPOLYSACCHARIDES
(EPS) MATRIX IN THE BIOCRUST
COMMUNITY

After cyanobacteria have been inoculated, biocrusts start to emerge along

with the development of an extracellular polymeric matrix (EPM) that is crucial to the
biocrust structure. The structural characteristics of cyanobacterial EPS vary depending
on the species and type of stress exposure, and they have a high average molecular mass.
Proteins, pigments, extracellular DNA, and reactive functional groups like sulphates and
phosphates are also present in EPS, which are primarily polysaccharidic in nature. The
availability of nutrients, the structure of the soil, and the size of the substrate all have a
significant impact on the physicochemical characteristics of EPS.

Figure 5.1: EPS-sheath collating close sand particles

Some of the beneficial roles of EPS for producing cyanobacterial cells and the
biocrust community are discussed below:

• Mediation of cell adhesion and cohesion : Depending on EPS physicochemical

properties, particularly composition and molecular weight distribution, EPS play
a key role in the production of microbiological aggregates and biocrusts.

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• Physical protection : EPS represent a physical barrier from external chemical and
physical harms including extreme temperatures, antibacterial agents, and predation
by protozoans.

• Control of cell water content : EPS counteract the loss of intracellular water by
reducing the entry of hypertonic ions into the cells and accumulate water-stress
proteins that maintain membrane stability.

• Protection from drought and strong light : Extracellular EPS structures, like the
sheath synthesized by M. vaginatus, protects filament bundles from drought, ex-
cessive sunlight and salt stress.

• Maintenance of soil moisture : EPS in the biocrust community reduce water infil-
tration and increase its permanence in the first soil layers.

• Water accumulation from non-rainfall sources : EPS amount in biocrusts is pos-

itively related to the ability to capture water from both rainfall, and non-rainfall
sources.

• Protection from UV radiation : The thicker the EPS envelope, the higher the
protection against UV radiation.

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Chapter 6
EVALUATION OF THE INOCULATION
EFFECT

By evaluating metrics linked to soil cyanobacterial colonisation, biomass growth,

and the properties of induced biocrusts, it is possible to determine the effects of cyanobac-
teria inoculation indirectly.[1].A thorough evaluation of the inoculation impact is possible
thanks to the set of analytical techniques listed in Table 6.1

Table 6.1: Set of analytical methods to assess inoculant establishment and growth in the
substrate, and the stability of induced biocrusts

Functional Parameters Index for

Chlorophyll a
EPS quantification
Total carbohydrate content
Reflectance
Biocrust thickness (BT)
Aggregate stability (AS)
Tensile strength (TS)
Threshold friction velocity (TFV)
Water drop penetration time test
Phototrophic state of biomass
Biocrust development
C soil pool and biocrust development
Substrate colonization and biocrust maturation
Biocrust maturation and biocrust level of development
Biocrust resistance to disaggregation by hydraulic forces
Biocrust resistance to penetration/ compression
Biocrust resistance to wind erosion
Persistence of hydrophobicity

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Chapter 7
FACTORS AFFECTING INOCULATION

The physiological traits of the biocrust species and their interactions, as well
as soil moisture, soil textural traits, light intensity, temperature, and nutrient availabil-
ity, can all affect the development of natural biocrusts in natural environments. These
factors are referred to as ”internal” and ”external,” respectively. The influence of these
characteristics must be taken into account when cyanobacteria are inoculated in order to
improve the success rate of the treatment.

Since mechanisms of adaptation to natural environment are species-specific,

the influence of several factors affecting the physiology of the inoculant and the formation
of biocrusts, are discussed below.

7.1 Soil salinity and pH

Cyanobacteria and biocrusts function as agents that lower soil salinity and
increase soil quality. Salinity is the total amount of dissolved inorganic ions, and it
can vary in composition as well as overall amount. Inorganic ions can be hazardous to
cyanobacteria, and high salt concentrations reduce water availability.Salinity, then, is a
factor that lowers the amount of cyanobacteria in some ecosystems. Soil pH is another
important parameter to consider [3]. Under natural conditions, most cyanobacteria grow
well on soils from neutral to alkaline, with the optimal pH ranging from 7.5 to 10 and
lower limits of 6.5–7.0.

7.2 Substrate Characteristics

Grain size and soil texture both have an impact on biocrust growth. Cre-
ating stable organo-mineral aggregates on the infected substrate is the first challenge
for cyanobacterial filaments. Both soil particle weight and interparticle cohesiveness are

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influenced by substrate particle size. Smaller sediments (in the silt and clay range) are
naturally stable because of chemical interactions, whereas larger particles (greater than
75-100 m) are more stable due to gravity. Sand grains intertwined in a network of fila-
ments with frequent air holes define biocrusts induced on sandy substrates. On silt loam
soils, by contrast, filament networks are denser and there are no air spaces. Because
voids prevent cyanobacteria from spreading, biocrusts grow more equally on fine surfaces
than on coarse ones.

7.3 Water Availability

The efficacy of the inoculation treatment depends on the availability of water.

Despite the established ability of cyanobacteria to tolerate drought conditions, restric-
tions in water supply can have an impact on the survival of the inoculant. Additionally,
water constraint has a deleterious impact on the growth of biocrust. Irrigation must be
used to support inoculation and the growth of biocrusts in the field. However, supplying
water can be expensive and technically difficult because it requires irrigation infrastruc-
ture, water transport, and water distribution equipment, particularly when dealing with
vast areas that are far from any research facilities or water reservoirs. The ability of
cyanobacteria to withstand drought varies depending on the species, but it may also be
influenced by the physicochemical properties of the soil.

7.4 Inoculum Concentration

An essential criterion for discrimination is inoculum concentration. The first

factor affecting inoculum survival and biocrust formation is inoculum concentration. Sec-
ond, the amount of biomass needed for each inoculation treatment affects cultivation ex-
penses and, consequently, the process’s economic balance, especially at large experimental
scales. Grams of dry biomass per square foot of substrate or grammes of chlorophyll per
substrate have been the two most common units used to express inoculum amounts. The
efficiency of the pigment extraction technique and species-specific inoculant properties
that may affect extraction yield can have an impact on the latter procedure whereas

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the former is more repeatable. Similar to how the weight of fresh biomass per area of
substrate when used to express inoculum yields approximations due to the residual water
content.

7.5 Temperature

Additionally to microbial numbers, metabolic activity, substrate absorption

rates, and community structure, temperature can have a substantial impact on soil
physicochemical properties. Due to the best growth of cyanobacterial species creating
biocrusts, the majority of laboratory-scale research have used incubator temperatures
between 25 and 30 °C. For cyanobacteria to adapt to unfavourable conditions and spread
across the soil in accordance with the temperature, physiological mechanisms are re-
quired.

7.6 Light Intensity

Cyanobacteria reside in the upper layers of soil because of their photosyn-

thetic metabolism.In research on inoculation, light levels during the tests ranged greatly
from 7 to 100 mol, and at least in one instance, inoculation was carried out in the pres-
ence of daylight. Different light levels cause temporal gene regulation, which in turn
triggers the expression of the glycogen synthesis and storage enzymes. The capacity of
the cyanobacteria to expand and colonise the substrate is confined, regardless of envi-
ronmental factors, when glycogen is mostly stored and not utilised for physiological and
catabolic functions.

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Chapter 8
IMPORTANCE OF BIOLOGICAL
CRUSTS

Biocrusts are tiny ecosystems that carry out all of a bigger ecosystem’s essen-
tial tasks, albeit on a smaller scale. Biocrusts provide disproportionately more ecosystem
benefits across scales than their biomass would suggest. Increased soil aggregation caused
by biocrusts lowers soil loss due to wind and water erosion. Together, biocrusts’ primary
producers fix 0.58 Pg C annually, and they also fix 24 Tg of nitrogen. The interaction be-
tween soil type and climate affects how biocrusts affect hydrology, sometimes increasing
run-off and sometimes enhancing infiltration.Biocrusts can operate as an armour, in-
hibiting seed penetration, but if seeds can get through, biocrusts can encourage seedling
establishment and growth as well as decreases in exotic plant cover. Biocrusts also inter-
act negatively and positively with plant establishment and growth. Additionally, it has
favourable impacts on soil food webs, supplying food and habitat to bacteria, fungus,
and micro-, meso-, and macrofauna as well as to their production, fertility, and other
ecological functions.

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Chapter 9
CONCLUSION

The inoculation of cyanobacteria is a potential approach that, if used, might

assist researchers in tackling land rehabilitation and reversing desertification. The appli-
cation of technology, which depends on closing the aforementioned knowledge gaps, will
not only serve as a tool to counteract land degradation, but it may also be used to address
a number of associated issues, such as dust storms and increased soil erodibility. In re-
gions of the world where the phenomena of desertification or soil deterioration is already
taking place, this technology could become a widely used tool, even by non-academics
(such as small farmers).

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REFERENCES

[1] Antonika A , Faist A , Rodriguez Caballero E , Young K.E , Chaudhary V.B ,

Condon L.A & Pyke D.A. “Biological soil crusts in ecological restoration : emerging
research and perspectives”. In: Restoration Ecology S3-S8.28 (2020).

[2] Chamizo S , Mugnai G , Rossi F, Certini G & De Philippis R. “Cyanobacteria

inoculation improves soil stability and fertility on different textured soil: gaining
insights for applicability in soil restoration”. In: Frontiers in Environmental Science
6.49 (2018).

[3] Kheirfarm H , Sadeghi S.H , Homaee M & Darki B.Z. “Quality improvement of an
erosion-prone soil through microbial enrichment”. In: Soil and Tillage Research 165
(2022).

[4] Rossi F , Mugnai G & De Philippis R. “Cyanobacterial biocrust induction: A com-

prehensive review on a soil rehabilitation-effective biotechnology”. In: Geoderma
415.115766 (2022).

[5] Xiao B , Bowker M.A , Zhao Y , Chamizo S & Issa O.M. “Biocrusts : Engineers and
architects of surface soil properties, functions, and processes in dryland ecosystems”.
In: Geoderma 424.116015 (2022).

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APPENDIX-1

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