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Development in
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Research and
Processes
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Development in
Wastewater Treatment
Research and
Processes
Microbial Degradation of
Xenobiotics Through Bacterial
and Fungal Approach
Edited by
Susana Rodriguez-Couto
Department of Separation Science, LUT School of
Engineering Science, LUT University, Mikkeli, Finland
Maulin P. Shah
Environmental Microbiology Lab, Bharuch, Gujarat, India
Elsevier
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Contents
Contributors ....................................................................................................................................... xvii
CHAPTER 1 Microbial degradation of xenobiotics in bioelectrochemical

systems.................................................................................................. 1
Somdipta Bagchi and Manaswini Behera
1.1 Introduction to xenobiotics .........................................................................................1
1.2 Conventional processes of xenobiotics degradation and their drawbacks.................3
1.2.1 Adsorption ........................................................................................................ 3
1.2.2 Membrane processes ........................................................................................ 4
1.2.3 Advanced oxidation processes ......................................................................... 4
1.2.4 Biological treatment processes......................................................................... 5
1.3 Xenobiotics degradation in bioelectrochemical systems ...........................................6
1.3.1 Microbial fuel cell ............................................................................................ 6
1.3.2 Microbial electrolysis cell .............................................................................. 11
1.3.3 Microbial desalination cell............................................................................. 14
1.4 Challenges faced .......................................................................................................15
1.5 Summary ...................................................................................................................15
1.6 Conclusion ................................................................................................................16
References.................................................................................................................16
Further reading .........................................................................................................22
CHAPTER 2 Bacterial and fungal degradation of dyes: a remedial source ......... 23
Bhumika Rajoria and Arpita Roy
2.1 Introduction.............................................................................................................23
2.2 Classification of dyes..............................................................................................24
2.3 Environmental and health problems associated with dyes ....................................25
2.4 Conventional methods of dye removal...................................................................26
2.5 Bacterial degradation of dye ..................................................................................28
2.5.1 Pure bacterial cultures for dye degradation .................................................28
2.5.2 Mixed cultures and cocultures for degradation ...........................................29
2.5.3 Immobilized cells for degradation ...............................................................29
2.5.4 Microbial fuel cells for degradation ............................................................30
2.6 Mechanism of dye degradation via bacteria ..........................................................30
2.6.1 Degradation via enzymes .............................................................................30
2.6.2 Degradation via mediation ...........................................................................32
2.6.3 Decolorization via organic and inorganic compounds ................................32
2.7 Factors affecting bacterial degradation of dyes .....................................................32
2.7.1 Aeration and agitation ..................................................................................32
v
vi Contents
2.7.2 Temperature ..................................................................................................33

2.7.3 pH .................................................................................................................33
2.7.4 Dye concentration.........................................................................................33
2.7.5 Dye structure ................................................................................................33
2.8 Bacterial bioreactors for decolorization and degradation of dyes.........................34
2.9 Fungal degradation of dye......................................................................................34
2.10 Mechanism of dye degradation via fungi...............................................................35
2.10.1 Bioaccumulation of dye .............................................................................35
2.10.2 Biosorption of dye......................................................................................36
2.10.3 Biodegradation of dye ................................................................................36
2.11 Factors affecting fungal degradation......................................................................37
2.11.1 Aeration and agitation ................................................................................37
2.11.2 Temperature ................................................................................................37
2.11.3 pH ...............................................................................................................38
2.11.4 Dye concentration.......................................................................................38
2.11.5 Dye structure ..............................................................................................38
2.12 Fungal bioreactors for decolorization and degradation of dyes ............................38
2.13 Conclusion and future prospects ............................................................................39
References...............................................................................................................40
Further reading .......................................................................................................43
CHAPTER 3 Role of halophiles in xenobiotic bioremediation .............................. 45
Arunima Bhattacharjee, Aveepsa Sengupta, Sanchayita Basu,
Pragati Singh and Ashutosh Kumar
3.1 Xenobiotic compounds .............................................................................................45
3.2 Halophilesdan extremophle in action .....................................................................46
3.3 Metal bioremediation by halophiles .........................................................................49
3.4 Bioremediation of hydrocarbons including crude oils.............................................50
3.5 Bioremediation of crude oil......................................................................................51
3.6 Aliphatic hydrocarbon ..............................................................................................52
3.7 Monoaromatic hydrocarbon......................................................................................52
3.8 Polyaromatic hydrocarbon........................................................................................54
3.9 Conclusion ................................................................................................................55
References.................................................................................................................55
Further reading .........................................................................................................60
CHAPTER 4 Fungal diversity in the bioremediation of toxic effluents ................. 61
Mohita Chugh, Lakhan Kumar and Navneeta Bharadvaja
4.1 Introduction...............................................................................................................61
4.2 Methods of bioremediation.......................................................................................62
4.3 Steps of mycoremediation ........................................................................................63
4.3.1 Biosorption ..................................................................................................... 63
4.3.2 Bioaccumulation............................................................................................. 64
Contents vii
4.3.3 Biodegradation................................................................................................ 64
4.3.4 Bioconversion ................................................................................................. 65
4.4 Kinds of bioremediators ...........................................................................................65
4.4.1 White-rot fungi ............................................................................................... 65
4.4.2 Marine fungi ................................................................................................... 68
4.4.3 Extremophilic fungi........................................................................................ 69
4.4.4 Symbiotic fungi .............................................................................................. 69
4.4.5 Other kinds of mushrooms ............................................................................. 69
4.5 Factors affecting mycoremediation ..........................................................................70
4.5.1 Temperature effect on mycoremediation ....................................................... 70
4.5.2 pH effect on mycoremediation....................................................................... 70
4.5.3 Relative humidity effect on mycoremediation .............................................. 71
4.5.4 Effect of oxygen, light, trace elements, and aeration on
mycoremediation ............................................................................................ 71
4.6 Different types of targeted contaminants .................................................................71
4.6.1 Volatile organic compounds ........................................................................... 71
4.6.2 Synthetic dyes................................................................................................. 73
4.6.3 Heavy metals .................................................................................................. 74
4.6.4 Toxic compounds and municipal solid wastes .............................................. 74
4.7 Enzymes involved in bioremediation .......................................................................75
4.7.1 Peroxidase....................................................................................................... 76
4.7.2 Laccase ........................................................................................................... 77
4.7.3 Cytochrome P450 ........................................................................................... 77
4.8 Recent advancements in fungal bioremediation ......................................................78
4.9 Conclusion and future perspective ...........................................................................79
References.................................................................................................................80
Further reading .........................................................................................................88
CHAPTER 5 Current advances in microbial bioremediation of surface
and ground water contaminated by hydrocarbon.............................. 89
Micheal Bukola Alao and Elijah Adegoke Adebayo
5.1 Introduction...............................................................................................................89
5.1.1 Hydrocarbon (petroleum) contamination of surface water and
ground water................................................................................................... 90
5.1.2 Management of hydrocarbon contaminated water ........................................ 92
5.2 Microbial bioremediation of hydrocarbon (petroleum) contaminated water ..........94
5.2.1 Advantages of microbial bioremediation technique ...................................... 96
5.2.2 Current advances in microbial bioremediation technique ............................. 97
5.2.3 Mechanisms of bioremediating activity by microorganisms....................... 102
5.2.4 Current advances in application of microbial bioremediation .................... 105
5.3 Challenges and recommendations ..........................................................................109
5.3.1 Challenges .................................................................................................... 109
viii Contents
5.3.2 Recommendations ........................................................................................ 111

5.4 Conclusions and future prospects...........................................................................111
References...............................................................................................................112
Further reading .......................................................................................................116
CHAPTER 6 Microbial remediation of petroleum hydrocarbons in liquid
wastes ............................................................................................... 117
Rahul Saha and Soma Nag
6.1 Introduction...........................................................................................................117
6.2 Classification and composition of petroleum oil .................................................118
6.2.1 Very light oils .............................................................................................118
6.2.2 Light oils.....................................................................................................119
6.2.3 Medium oils................................................................................................119
6.2.4 Heavy fuel oils ...........................................................................................119
6.3 Impact of petroleum hydrocarbons on environment............................................119
6.4 Remediation techniques........................................................................................119
6.4.1 Physical techniques ....................................................................................120
6.4.2 Chemical techniques ..................................................................................120
6.4.3 Bioremediation techniques .........................................................................120
6.4.4 Phytoremediation........................................................................................120
6.5 Microbial degradation petroleum oil....................................................................121
6.6 Sources of microbes .............................................................................................122
6.7 Role of enzymes ...................................................................................................123
6.8 Role of biosurfactants in degrading hydrocarbon................................................124
6.9 Factors affecting the biodegradation of petroleum hydrocarbon.........................125
6.10 Advantages of microbial remediation technique .................................................125
6.11 Conclusion and future prospects ..........................................................................125
References.............................................................................................................126
Further reading .....................................................................................................129
CHAPTER 7 Microbial remediation of metals by marine bacteria ..................... 131
Sougata Ghosh, Joorie Bhattacharya, Rahul Nitnavare and
Thomas J. Webster
7.1 Introduction.............................................................................................................131
7.2 Heavy metal toxicity...............................................................................................132
7.3 Removal of toxic metals by marine microbes .......................................................134
7.3.1 Arsenic........................................................................................................ 134
7.3.2 Cadmium .................................................................................................... 134
7.3.3 Chromium ................................................................................................... 138
7.3.4 Cobalt.......................................................................................................... 138
7.3.5 Copper ........................................................................................................ 139
7.3.6 Iron.............................................................................................................. 141
Contents ix
7.3.7 Lead ............................................................................................................ 142

7.3.8 Manganese .................................................................................................. 145
7.3.9 Mercury ...................................................................................................... 146
7.3.10 Nickel.......................................................................................................... 148
7.3.11 Selenium ..................................................................................................... 149
7.3.12 Vanadium .................................................................................................... 151
7.3.13 Zinc ............................................................................................................. 152
7.4 Conclusions and future perspectives ......................................................................153
Acknowledgments...................................................................................................154
References...............................................................................................................154
Further reading .......................................................................................................158
CHAPTER 8 Microbial degradation of dye-containing wastewater .................... 159
Kumari Guddi and Angana Sarkar
8.1 Introduction...........................................................................................................159
8.2 Dye........................................................................................................................160
8.3 Classification of dyes............................................................................................160
8.4 Sources of hazardous dyes in the environment....................................................161
8.5 Mode of toxicity of dye in human health ............................................................164
8.6 Microbial interaction with dyes ...........................................................................165
8.7 Bacterial interaction with dye ..............................................................................166
8.8 Fungal interaction with dye..................................................................................166
8.9 Bioremediation strategies of dyes ........................................................................167
8.10 Bacterial degradation of dyes...............................................................................167
8.11 Fungal degradation of dyes ..................................................................................170
8.12 Hurdles on dye bioremediation ............................................................................171
8.13 Future prospects....................................................................................................172
References.............................................................................................................172
Further reading .....................................................................................................175
CHAPTER 9 Microbial bioremediation of heavy metals by Marine bacteria ..... 177
Raja Husain, Nitin Vikram, Garima Yadav, Deepak Kumar,
Sonika Pandey, Monika Patel, N.A. Khan and Touseef Hussain
9.1 Introduction...........................................................................................................177
9.2 Types of marine bacteria involved in metal bioremediation ...............................179
9.2.1 Diversity of marine bacteria.......................................................................180
9.3 Mechanism of microbial bioremediation of metals .............................................181
9.3.1 Remediation and metals .............................................................................181
9.4 Microbial bioremediation and its mechanism......................................................182
9.5 Microbial remediation of heavy metals from the soil .........................................183
9.6 Microbial bioremediation of metals from water..................................................184
9.7 Advantages and disadvantages of microbial bioremediation ..............................185
x Contents
9.8 Bioleaching (biomining).......................................................................................186

9.9 Phytoremediation ..................................................................................................187
9.10 Plant microbial bioremediation ............................................................................188
9.11 Heavy metal removal by fungi.............................................................................189
9.12 Metal uptake by fungi ..........................................................................................191
9.13 Heavy metal removal by Algae............................................................................193
9.14 Factors affecting heavy metal bioremediation by algae ......................................194
9.15 Factors affecting the microbial bioremediation of heavy metals ........................195
9.15.1 Physiochemical factors affecting bioremediation....................................196
9.15.2 Biological factors affecting bioremediation processes ............................196
9.15.3 Climate change and bioremediation processes........................................196
9.16 Conclusion and Future prospects .........................................................................196
Acknowledgment ..................................................................................................197
References.............................................................................................................197
Further reading .....................................................................................................203
CHAPTER 10 Role of microbes in biodegradation of cyanide and its metal
complexes ....................................................................................... 205
Vandita Anand and Anjana Pandey
10.1 Introduction...........................................................................................................205
10.2 Cyanide .................................................................................................................206
10.2.1 Chemistry of cyanide and their various forms ........................................206
10.2.2 Sources of cyanide ...................................................................................208
10.2.3 Cyanide discharge from industries and pollution ....................................209
10.2.4 Toxicological effects of cyanide ..............................................................211
10.2.5 Effluent disposal standards of cyanide.....................................................213
10.3 Factors responsible for the biodegradation of cyanides ......................................213
10.3.1 Concentration of cyanides........................................................................214
10.3.2 Availability of nutrients............................................................................214
10.3.3 Availability of oxygen ..............................................................................214
10.3.4 Presence of additional pollutants .............................................................215
10.3.5 Temperature and pH .................................................................................215
10.4 Microbial metabolism...........................................................................................215
10.4.1 Hydrolytic pathway ..................................................................................216
10.4.2 Oxidative pathway....................................................................................217
10.4.3 Reductive pathway ...................................................................................218
10.4.4 Substitution/transfer pathway...................................................................219
10.4.5 Synthases pathways ..................................................................................219
10.5 Advances in cyanide biodegradation technologies ..............................................220
10.6 Conclusion and future prospects ..........................................................................221
References.............................................................................................................222
Further reading .....................................................................................................224
Contents xi
CHAPTER 11 Microbial-mediated explosives removal and its impact

on TNT, RDX, and HMX ................................................................... 225
Komal Agrawal and Pradeep Verma
11.1 Introduction...........................................................................................................225
11.2 Classification of explosives ..................................................................................226
11.3 The problem with explosives ...............................................................................227
11.4 Impact of explosives on the environment ............................................................227
11.5 An overview of the environmental fate of explosives .........................................228
11.6 Bioremediation of explosives ...............................................................................229
11.6.1 TNT ..........................................................................................................229
11.6.2 RDX..........................................................................................................231
11.6.3 HMX .........................................................................................................233
11.7 Limitations, future prospects, and conclusion .....................................................234
References.............................................................................................................235
Further reading .....................................................................................................241
CHAPTER 12 Advancement in microbial bioremediation.................................... 243
Dixita Chettri, Bhaskar Sharma and Anil Kumar Verma
12.1 Introduction...........................................................................................................243
12.2 Xenobiotics and role of microbes ........................................................................244
12.2.1 Biodegradation..........................................................................................245
12.2.2 Biotransformation.....................................................................................245
12.2.3 Cometabolism...........................................................................................245
12.3 Parameters for biodegradation..............................................................................245
12.3.1 Microbial structure ...................................................................................246
12.3.2 Environmental factors ..............................................................................247
12.3.3 Physiochemical characteristic of the contaminant ..................................247
12.4 Approaches to bioremediation..............................................................................248
12.4.1 Cell-based bioremediation........................................................................248
12.4.2 Enzyme-based bioremediation .................................................................250
12.5 Improving the process of biodegradation.............................................................251
12.5.1 Genetic engineering..................................................................................251
12.5.2 Biosurfactants ...........................................................................................254
12.5.3 Chemotaxis and transport.........................................................................254
12.6 Omics approaches in the microbial bioremediation ............................................255
12.7 Conclusion and future prospective .......................................................................258
Acknowledgment ..................................................................................................258
References.............................................................................................................259
Further reading .....................................................................................................262
xii Contents
CHAPTER 13 Counterbalancing common explosive pollutants (TNT, RDX,

and HMX) in the environment by microbial degradation.............. 263
Nikita Chakraborty, Pakiza Begum and Bhisma Kumar Patel
13.1 Introduction...........................................................................................................263
13.1.1 Biological remediation .............................................................................264
13.1.2 Explosives.................................................................................................265
13.2 TNT.......................................................................................................................266
13.2.1 Chemistry and properties of TNT............................................................266
13.2.2 Worldwide deposits of TNT.....................................................................267
13.2.3 Toxicity of TNT .......................................................................................269
13.2.4 Microbial degradation of TNT.................................................................270
13.3 RDX and HMX.....................................................................................................286
13.3.1 Chemistry and properties of RDX and HMX..........................................286
13.3.2 Toxicity of RDX and HMX .....................................................................287
13.3.3 Biodegradation of RDX and HMX ..........................................................288
13.4 Conclusion ............................................................................................................301
13.5 Future research .....................................................................................................303
References.............................................................................................................304
CHAPTER 14 Enzyme-based biodegradation of toxic environmental
pollutants ........................................................................................ 311
Nahid Masood Siddiqui and Praveen Dahiya
14.1 Introduction...........................................................................................................311
14.2 Structure of enzymes ............................................................................................313
14.3 Mode of action of enzymes ..................................................................................314
14.3.1 Fisher template model (lock and key model) ..........................................314
14.3.2 Induced fit model......................................................................................314
14.4 Classification and nomenclature...........................................................................315
14.4.1 Oxidoreductase .........................................................................................315
14.4.2 Transferase................................................................................................316
14.4.3 Hydrolases ................................................................................................317
14.4.4 Lyases .......................................................................................................318
14.4.5 Isomerases ................................................................................................318
14.4.6 Ligases ......................................................................................................319
14.5 Industrial pollution and their impact on the ecosystem.......................................320
14.5.1 Water pollution .......................................................................................320
14.5.2 Air pollution ...........................................................................................321
14.5.3 Soil pollution ..........................................................................................322
14.5.4 Effects on plants .....................................................................................323
14.5.5 Effect on wildlife....................................................................................323
Contents xiii
14.5.6 Enzymes-based biodegradation of various industrial pollutants ...........324

14.5.7 Enzyme oxidoreductases ........................................................................324
14.5.8 Enzyme peroxidases ...............................................................................325
14.5.9 Enzyme laccases.....................................................................................327
14.5.10 Enzyme hydrolases.................................................................................327
14.5.11 Limitations and recent advances ............................................................328
14.6 Conclusion ............................................................................................................330
References.............................................................................................................330
Further reading .....................................................................................................333
CHAPTER 15 Cyanoremediation: a clean and green approach toward the
sustainable environment ................................................................ 335
Subhasish Dutta, Debojit Ghosh, Arunima Lahiri,
Sourish Chakraborty and Soumya Pandit
15.1 Introduction...........................................................................................................335
15.2 Cyanobacteria at a glance.....................................................................................338
15.2.1 Microbiology of cyanobacteria ................................................................338
15.3 Cyanobacteria as potential food, feed, and bioenergy source .............................338
15.4 Cyanoremediationda clean and green technology toward sustainable
future .....................................................................................................................339
15.4.1 Characteristics of cyanobacteria ..............................................................340
15.4.2 Different cyanoremediation approaches ..................................................340
15.5 Conclusion ............................................................................................................349
References.............................................................................................................350
Further reading .....................................................................................................354
CHAPTER 16 Enzymatic bioremediation: current status, challenges, future
prospects, and applications........................................................... 355
Luciana Porto de Souza Vandenberghe, Nelson Libardi Junior,
Kim Kley Valladares-Diestra, Susan Grace Karp,
Joyce Gueiros Wanderley Siqueira, Cristine Rodrigues and
Carlos Ricardo Soccol
16.1 Introduction...........................................................................................................355
16.2 Bioremediationdaspects and techniques.............................................................355
16.3 Enzymes for bioremediation ................................................................................358
16.3.1 Oxidoreductases........................................................................................358
16.3.2 Hydrolases ................................................................................................362
16.4 Modified enzymes for bioremediationdgenetic engineering/enzyme
engineering............................................................................................................367
16.5 Enzyme immobilization........................................................................................369
16.6 Advances on an innovation on enzymatic bioremediation ..................................371
xiv Contents
16.6.1 Nanozymes ...............................................................................................371

16.6.2 Classification of nanozymes.....................................................................372
16.6.3 Nanozymes applied in bioremediation processes ....................................373
16.6.4 Future prospects .......................................................................................374
References.............................................................................................................374
CHAPTER 17 An approach toward the biodegradation of PAHs by microbial
consortia ......................................................................................... 383
Subhasish Dutta and Sayan Laha
17.1 Introduction...........................................................................................................383
17.2 PAH-degrading pathways by different bacteria and fungi...................................385
17.2.1 Degradation mechanism of fungi.............................................................385
17.2.2 Nonligninolytic fungi mechanism............................................................389
17.2.3 PAHs degradation pathway by bacteria ...................................................392
17.3 Removal of PAHs from the soil by bacteria ........................................................395
17.3.1 Removal of some PAHs from soil ...........................................................395
17.3.2 Fenton’s reaction with viable cells ..........................................................395
17.3.3 Biostimulation and bioaugmentation .......................................................396
17.3.4 Remediation mechanism ..........................................................................396
17.3.5 Competition with microflora in contaminated soil..................................397
17.3.6 Competition ..............................................................................................398
17.4 Effect of incomplete combustion of PAHs on microbial and fungal
consortia................................................................................................................398
17.5 Proteomics and metabolomics in bioremediation of PAHs .................................399
17.6 Limiting factors of PAHs degradation .................................................................400
17.7 Remediation strategy for limiting factors ............................................................400
17.7.1 Choice of microorganism .........................................................................400
17.7.2 Bioavailability issue .................................................................................401
17.7.3 Other factors .............................................................................................402
17.8 Review on physicochemical treatments ...............................................................402
17.9 Conclusion and future aspects..............................................................................402
References.............................................................................................................403
Further reading .....................................................................................................406
CHAPTER 18 Bacterial- and fungal-mediated biodegradation of petroleum
hydrocarbons in soil....................................................................... 407
Vivek Kumar Gaur, Varsha Tripathi and Natesan Manickam
18.1 Introduction...........................................................................................................407
18.2 Composition of petroleum hydrocarbons.............................................................410
18.2.1 Aliphatics..................................................................................................411
18.2.2 Aromatics..................................................................................................412
Contents xv
18.2.3 Asphaltenes and waxes.............................................................................413

18.3 Impact of petroleum hydrocarbon on soil............................................................415
18.3.1 Soil fertility...............................................................................................415
18.3.2 Soil microflora ..........................................................................................415
18.4 Removal of petroleum hydrocarbon.....................................................................417
18.4.1 Bacterial biodegradation of petroleum hydrocarbon ...............................417
18.4.2 Fungal biodegradation of petroleum hydrocarbons .................................419
18.5 Conclusions...........................................................................................................421
Acknowledgments.................................................................................................421
References.............................................................................................................421
CHAPTER 19 Deep-marine bacteriadThe Frontier alternative for
heavy metals bioremediation ......................................................... 429
Neha Singh, Umesh Goutam and Mrinmoy Ghosh
19.1 Introduction...........................................................................................................429
19.2 Sources of heavy metals.......................................................................................431
19.2.1 Toxic effects of heavy metals on sea life ................................................432
19.3 Characteristics and diversity of deep-marine bacteria.........................................433
19.4 Deep-marine bacteria adaptation concerning changing environmental
conditions..............................................................................................................434
19.4.1 Factors affecting bioremediation process ................................................435
19.4.2 Mechanism of remediation processes ......................................................436
19.5 Heavy metal removal by deep-marine bacteria ...................................................439
19.6 Application of deep-marine bacteria in bioremediation ......................................440
19.7 Bioremediation enhancement via genetically modified deep-marine
bacteria..................................................................................................................441
19.7.1 Nickel........................................................................................................443
19.7.2 Cadmium ..................................................................................................443
19.7.3 Arsenic......................................................................................................443
19.7.4 Mercury ....................................................................................................444
19.8 Future prospective also pros and cons of using deep-marine bacteria................444
19.9 Conclusion ............................................................................................................446
References.............................................................................................................446
Further reading .....................................................................................................450
CHAPTER 20 Microbial biofilms for waste treatment and sustainable
development.................................................................................... 451
Arunima Bhattacharjee, Priya Khadgawat, Abhinav Suresh, Jolly Thomas,
Gayatri Brahmandam, Pragati Singh and Ashutosh Kumar
20.1 Introduction...........................................................................................................451
20.2 Biofilms in bioremediation...................................................................................452
xvi Contents
20.3 Oil bioremediation................................................................................................455

20.4 Textile wastewater treatment ................................................................................456
20.5 Removal of pharmaceuticals ................................................................................458
20.6 Bioremediation of persistent organic pollutants ..................................................458
20.7 Heavy metal remediation......................................................................................460
20.8 Limitations and future prospect ...........................................................................461
20.9 Conclusion ............................................................................................................461
References.............................................................................................................462
Further reading .....................................................................................................465
Index ................................................................................................................................................. 467

Contributors
Elijah Adegoke Adebayo
Department of Pure and Applied Biology, Microbiology Unit, Ladoke Akintola
University of Technology, Ogbomoso, Oyo, Nigeria
Komal Agrawal
Bioprocess and Bioenergy Laboratory, Department of Microbiology, Central
University of Rajasthan, Ajmer, Rajasthan, India
Micheal Bukola Alao
Department of Pure and Applied Biology, Microbiology Unit, Ladoke Akintola
University of Technology, Ogbomoso, Oyo, Nigeria
Vandita Anand
Department of Biotechnology, Motilal Nehru National Institute of Technology
(MNNIT) Allahabad, Prayagraj, Uttar Pradesh, India
Somdipta Bagchi
School of Infrastructure, Indian Institute of Technology Bhubaneswar,
Bhubaneswar, Odisha, India
Sanchayita Basu
Department of Microbiology, Tripura University (A Central University), Agartala,
Tripura, India
Pakiza Begum
Department of Chemistry, Gargaon College, Gargaon, Assam, India
Manaswini Behera
School of Infrastructure, Indian Institute of Technology Bhubaneswar,
Bhubaneswar, Odisha, India
Navneeta Bharadvaja
Department of Biotechnology, Delhi Technological University, Delhi, India
Arunima Bhattacharjee
Tripura, India
Joorie Bhattacharya
Genetic Gains, International Crops Research Institute for the Semi-Arid Tropics,
Hyderabad, Telangana, India
Gayatri Brahmandam
Sreenidhi Institute of Science and Technology, Hyderabad, Telangana, India
Nikita Chakraborty
Department of Chemistry, Indian Institute of Technology Guwahati, North
Guwahati, Assam, India
xvii
xviii Contributors
Sourish Chakraborty
Department of Biotechnology, Haldia Institute of Technology (HIT), ICARE
Complex, Haldia, West Bengal, India
Dixita Chettri
Department of Microbiology, Sikkim University, Gangtok, Sikkim, India
Mohita Chugh
Praveen Dahiya
Amity Institute of Biotechnology, Amity University Uttar Pradesh (AUUP), Noida,
India
Subhasish Dutta
Complex, Haldia, West Bengal, India; Chemical Engineering Division, Center of
Innovative and Applied Bioprocessing (CIAB), Mohali, Punjab, India
Vivek Kumar Gaur
Environmental Biotechnology Laboratory, Environmental Toxicology Group, CSIR-
Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India; Amity
Institute of Biotechnology, Amity University Uttar Pradesh, Lucknow Campus,
Lucknow, Uttar Pradesh, India
Debojit Ghosh
Mrinmoy Ghosh
KIIT-Technology Business Incubator, Kalinga Institute of Industrial Technology
University, Bhubaneswar, Orissa, India
Sougata Ghosh
Department of Microbiology, School of Science, RK University, Rajkot, Gujarat,
India; Department of Chemical Engineering, Northeastern University, Boston,
MA, United States
Umesh Goutam
Department of Genetics Engineering and Molecular Biology, School of
Bioengineering and Biosciences, Lovely Professional University, Phagwara,
Punjab, India
Kumari Guddi
National Institute of Technology, Rourkela, Odisha, India
Contributors xix
Raja Husain
Department of Agriculture, Himalayan University, Itanagar, Arunachal Pradesh,
India; ICAR-Indian Institute of Pulses Research, Kanpur, Uttar Pradesh, India;
Department of Plant Molecular Biology and Genetic Engineering, Narendra Deva
University of Agriculture and Technology, Ayodhya, Uttar Pradesh, India
Touseef Hussain
Department of Botany, Aligarh Muslim University, Aligarh, Uttar Pradesh, India;
Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New
Delhi, Delhi, India
Nelson Libardi Junior
Departamento de Engenharia de Bioprocessos e Biotecnologia, Universidade
Federal do Paraná e UFPR, Curitiba, Parana, Brazil
Susan Grace Karp
Departamento de Engenharia de Bioprocessos e Biotecnologia, Universidade
Federal do Paraná e UFPR, Curitiba, Parana, Brazil
Priya Khadgawat
Department of Genetics, University of Delhi, New Delhi, Delhi, India
N.A. Khan
Department of Plant Molecular Biology and Genetic Engineering, Narendra Deva
University of Agriculture and Technology, Ayodhya, Uttar Pradesh, India
Ashutosh Kumar
Tripura, India
Deepak Kumar
Department of Manufacturing & Development, Nextnode Bioscience Pvt. Ltd.,
Kadi, Gujarat, India; Department of Plant Molecular Biology and Genetic
Engineering, Narendra Deva University of Agriculture and Technology, Ayodhya,
Uttar Pradesh, India
Lakhan Kumar
Sayan Laha
Arunima Lahiri
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All at once the negro, with a roar like that of a wild beast, whipped a
curved sword out of a belt about his waist, and, springing forward, delivered
a furious sweeping cut which, if it had taken effect, must have severed
Royce's head from his shoulders.
Fortunately for him, however, he was quick of eye and wit, and nimble in
his movements. At school he had had no match in boxing and fencing. Being
absolutely unarmed, he had no means of parrying the stroke; but he dropped
on one knee, and the scimitar whistled within an inch of his crown, striking
with a crashing stroke the wall on his right hand.
While the negro was still bent forward with the force of his blow, Royce
sprang low at his knees, and, tugging them towards him, brought the man
with a thud to the floor. The sword fell from his hand and clashed on the
stone flags, and Royce reached down to get hold of it. But the negro sprang
to his feet with agility amazing in so huge a man, and hurled himself upon
the Englishman.
Royce had just time to straighten himself and prevent himself from being
thrown down; the next moment the negro's arms were about him; he felt hot
breath upon his face, and saw the gleaming teeth and infuriated eyes of a
man from whom he knew he could expect no mercy.
He was well acquainted with the styles of wrestling in vogue in England

—the Cumberland, the Devon, the Lancashire; but he was instantly aware
that the negro's method was none of these. It was, in fact, a form of wrestling
like that which had been practised ages ago in the Olympic games, and had
no doubt been introduced into Northern Africa by the Romans in the days of
Cæsar and Pompey. It resembled the catch-as-catch-can style of Lancashire
more nearly than the lighter styles with which Royce was familiar.
The negro's aim seemed to be to throttle his opponent, or to squeeze the

breath out of his body; and Royce, struggle as he might, felt the thick,
muscular arms gripping him more and more closely. Slighter in build, he had
no chance of employing the feints and tricks which might have compensated
for a less powerful physique in dealing with an Englishman. In that straining
grasp, there was no hope for the lesser man; in a few seconds the struggle
must end.
AT GRIPS WITH THE NEGRO
The encounter, the coming to grips, had happened so swiftly that Royce
had had no time to think that there was help at hand in the shape of John. But
now, at this critical moment, when he felt that the very life was being
crushed out of him, he remembered the staunch companion of his journey,
who could not be far away.
Making a desperate effort to fill his lungs, he uttered a shout, or rather a

choking gurgle, which no one would have recognised as the voice of an
Englishman. The negro laughed, anticipating the moment when the white
man would lie limp and lifeless at his feet. Bub John, climbing the hill with a
rabbit dangling in his hand, heard the two sounds—the gasping cry, the loud,
mocking laugh. Hastening forward at a run, he shouted aloud, giving a long,
penetrating note like the yodel of the Swiss mountain shepherd. The sound,
growing louder moment by moment, came to the ears of the negro. He
realised instantly that, unless he could dispose of the Englishman at once, he
would soon have two men to deal with.
The encouraging sound gave Royce new strength. He put forth his last
energies to resist the strangling grip.
"Yoi-aloo! Yoi-aloo!"
The newcomer was close at hand. The panting negro lowered his arms,
caught Royce about the hips, and tried to lift him, intending to dash him
upon the floor. Royce flung his legs about the giant's thighs, stiffened his
muscles, and dragged with all his force upon the negro's shoulders.
"Yoi-aloo! Yoi-aloo!"
The game was up! The negro dared not wait longer. Loosening his grip,
he wrenched himself out of Royce's entwining arms, thrust him away, and,
turning about, rushed through the passage into the open. There he saw John
hurrying up within twenty paces of him, and swerving to the left, in five
seconds had disappeared among the bushes.
CHAPTER III
THE STORY OF GORUBA
Royce, when the negro left him, was breathless from the struggle. But he
had the presence of mind to run back to the room where he had passed the
night, pick up his rifle and revolver from the floor, and hurry to the entrance.
There John met him. The anxious alarm on the Hausa's face gave way to a
broad smile when he saw that his master was safe.
"Where is that ruffian?" asked Royce, looking round for his assailant.
"Him run away quick," replied John, pointing to the bushes. "Berry much
'fraid of me."
"You were just in time, John. He was crushing me to a jelly. It's a lesson
to me not to part with my revolver for an instant. You saw no other black
men about?"
"No, sah. Him berry big chap, sure 'nuff."
"A giant! I am bruised all over. I met him as I was coming out for a bathe,
and he sprang at me at once. Why should he do that?"
"Bad fella, sah."
"I daresay; but it was something more than original sin. He went for me
with as much ferocity as an eagle whose nest I disturbed once. Does this
place belong to him, I wonder?"
John could only repeat that the man was a "bad fella." But Royce felt a
good deal puzzled. The negro's savage onslaught might be explained by his
regarding the white man as a robber, but there appeared to be nothing in the
place worth stealing. It was strange that he should have so fiercely resented
what was, after all, an innocent intrusion.
"We'll have another look round before we start," said Royce. "Or, rather, I
will. You keep guard at the doorway, John, and call me if you see anyone
moving about outside."
Royce searched the building thoroughly. The result confirmed his
overnight impression, that it was in a fair state of preservation. But there was
nothing in any of the rooms to indicate present or even remote occupation.
Except for fragments of stone and rubble, they were bare. There was nothing
to tempt a robber. Royce could only conclude that the man had attacked him
from an instinct of self-preservation. What had led him to enter the building
was a mystery.
Royce returned to John, who during his absence had kindled a fire,
skinned the rabbit, and set it to roast. They made a good breakfast, then
started in the direction of the village where Royce hoped to purchase food
for his men.
"We must keep a good look-out," he said, "in case that fellow should be
one of a band prowling about here. He won't be difficult to recognise. There
can't be many men of his height and size. And if there were, I should know
him again by some strange marks on his face. Why do these black men gash
themselves, John?"
"To make him look pretty, sah."
"Um! They've a queer notion of beauty, then."
Anxious to accomplish his errand and return to Challis, Royce pushed on

as rapidly as possible. The country was pathless, for the most part flat, with
undulations here and there, covered with thick bush varied by an occasional
gum-tree. Drysdale's rough sketch-map gave him little more than a bare
direction, and he had to trust a good deal to luck. After three hours' steady
marching, which ought to have brought them to the village, if the estimate of
its distance were correct, they were still in the same wild, barren country,
without a sign of mankind. It seemed probable that they had overshot the
mark, so, after taking a short rest, they altered their direction in the hope of
discovering a path.
It was late in the afternoon, and they were very tired, when at last they
struck into a narrow, beaten track, far to the left of their original course.
"This looks promising," said Royce. "We'll make a spurt, John."

Another half-hour brought them to cultivated fields. Crossing these, they
found themselves faced by a mud wall, fourteen or fifteen feet high, pierced
by a single gate. This was closed.
"It's a town, after all," said Royce; "not a village. So much the better, if
the people are friendly. Give them a call, John."
JOHN ADDRESSES THE SENTRY
The Hausa let out his long yodelling cry. A head was seen peering over
the top of the wall. John lifted both hands, and spread them, palms open, as a
sign of friendliness; then began an address in the native tongue, somewhat as
follows:
"Ho, you admirable watchman of a noble chief! Tell your kind and
worthy lord that a stranger from the lands of the Great White King seeks to
look upon his face, and have a friendly talk with him. My lord and master is
a very great man, with horses and cattle in number as the grains in a
cornfield, and it is a great honour that he does to your chief. Open, then,
your gate, and let this great lord enter; and, as he passes, let your people fall
to the ground, and throw earth upon their bodies, as befits folk who are but
as worms in his sight."
Unconscious of the extravagant claims made on his behalf, Royce leant

on his rifle, waiting. The head disappeared. Some minutes elapsed; then the
watchman came back to his perch, and a long conversation ensued between
him and the Hausa, who grew more and more excited, and raised his voice
until it became almost a yell.
"What is it all about?" asked Royce, beginning to suspect that his man
was growing impolite.
"Son of a dog, dat fella!" replied John indignantly. "He want to know too
much. Talk 'bout sah's father and mother, how many wives, what he come
for, too much!"
"Did you tell him I want to buy food?"
"No, sah; I tell him——"
"Then do so at once," Royce interrupted sternly.
In a more subdued tone of voice, John gave his message. The man again
withdrew.
After another interval, the gate was thrown open, and Royce, entering,
found himself among a band of stalwart natives, carrying long, broad-bladed
spears, and marked on each cheek, near the ear, with five or six narrow cuts,
the badge of their tribe. Escorted by them, and watched by a curious crowd
of townsfolk, Royce proceeded to the chief's large mud house in the centre
of the town.
On entering he was greeted with the words: "Sanu, bature!" (Hail, white
man!) from a stout, pleasant-faced, bearded black man, somewhat past
middle age, who motioned to him to be seated, and ordered one of his slaves
to bring forward a present of a roasted fowl.
The customary salutations passed between host and visitor. Royce had
already learnt to endure this lengthy ceremony with patience. It was
something like this:
John: "Hail, chief! Is it well with you?"
Chief: "It is well."
John: "Allah be praised! Is it well with your wives?"
John: "Allah be praised! Is it well with your children?"
John: "Allah be praised! And your horses?"
John: "Allah be praised! And your cattle?"
Chief: "They are well."
John: "Allah be praised! Your house and all that is yours?"
Chief: "All is well."
John: "Allah be praised!"

And then they got to business. The chief apologised for the delay in
opening the gate. His watchmen had to be careful, because it was reported
that Tubus were in the neighbourhood. A few days before a fugitive had
come in from the westward, and told how his village had been raided and
destroyed. From the chief's description, Royce gathered that this was the
village which he had himself seen, and near which Challis was encamped.
Royce then explained the object of his visit, instructing John to translate
exactly what he said, without adding or subtracting anything. The chief at
once agreed to sell a quantity of millet and manioc for the men, and to
present Royce with a few dozen eggs—an offer which Royce gracefully
accepted, though he knew that most of the eggs would turn out to be bad.
The African native can never understand the white man's squeamishness in
the matter of addled eggs.
The next question was about the transportation of the food to the camp,
nearly twenty miles distant. At first the chief, for fear of the Tubus, was
reluctant to supply carriers. But when Royce explained that there had been
no sign of Tubus on the way, and that the area of their depredations appeared
to be considerably to the west, he yielded, and gave orders for the food to be
loaded into calabashes, and for a dozen slaves to be ready to start with them
in the morning.
It was clearly too late for a start to be made that evening, though Royce
was very anxious to get back to his friend. He accepted the chief's hospitality
for the night, and, though very tired, kept up a tedious conversation with him
through John. In the course of this he related the incident of the early
morning.
The chief seemed amused at the thought of a wrestling match between an

Englishman and a negro, and laughed heartily at the negro running away on
hearing John's cry, "Yoi-aloo!" which he made the Hausa repeat again and
again. But his amusement soon gave place to alarm, his smiles to a look of
thoughtfulness. He had asked Royce to describe the negro. He seemed little
impressed by details of the man's height and size, but when Royce
mentioned that he had noticed two straight cuts down the middle of each
cheek he uttered a sudden, sharp exclamation.
"Goruba!"
"What does he mean by Goruba?" asked Royce of John, after a brief

silence.
The chief seemed to deliberate whether he should speak or not. At last he

said:
"I will tell the white man—my friend. Years ago I was chief of only a
small village, and lord of little wealth. And I sought to increase my wealth
by prudent trading, to which end I hunted the elephant, and sold his tusks to
merchants from the East. And one time, having got together some few tusks
in readiness for the barter, I was beset in my village by a horde of strange
warriors, armed with guns, a terror to all my people. And these evil-doers
came to me and made me captive, and demanded that I should deliver up to
them those few tusks which were the spoil of my hunting. And, when I
refused, they treated me shamefully and cruelly, so that I bear the marks on
my body to this day. And there was no help for me, no hope of deliverance;
and then, for the safety of my life, I was fain to yield, and saw myself robbed
of the treasure that had fallen to my spear.
"And the captain of those wretches, he that put me to the torture, was a
man of vast stature and the strength of a giant, Goruba by name. He was
from the east country, the slave of Rabeh, King of Dar Runga, who laid
waste all the lands on this side of the great lake, and whose warriors were as
locusts on the face of the earth.
"It was a good day for all this country when Rabeh was slain, and his men
were scattered to the winds. I knew not what had become of Goruba, one of
his chiefest captains; but in very truth it was he that laid hands on you, for
his stature and those marks upon his cheeks betray him. And this news that
you give me is heavy upon my heart, for without doubt Goruba is again
prowling like a lion about these lands, and many a village will fall a prey to
him."
Royce did his best to reassure the chief, pointing out that Goruba had
been alone, and there was no evidence that he was the leader of any
considerable body of men. This somewhat heartened the old man, who
declared, however, that he would henceforth be doubly watchful, and
advised the white man to leave the country as soon as possible.
"I shall go at my own time," said Royce quietly. "I thank you for your
warning, and will do what I can to repay your kindness."
Next morning he left the village with John and a dozen carriers, well
loaded with food-stuffs, and hastened at his best pace to rejoin his friend.
CHAPTER IV
RUSHED BY TUBUS
Challis, after Royce had left him, felt somewhat uneasy. Royce had
spoken hopefully of getting back on the following day, but Challis,
remembering the difficulties of finding the way in a strange country, was
pretty sure that Royce had overestimated his powers, and was prepared to be
left in sole charge for at least two days, and perhaps three. It was not a
pleasant prospect, for the raiders could not be very far away, and the
possibility of being attacked was disturbing. He kept a smiling face,
however, and said nothing about his anxieties to the boys.
The first thing to do was to form as secure a camp as possible. The

camping arrangements had been interrupted by the discussion leading to
Royce's departure. As soon as Royce was gone, Challis took the matter in
hand.
The spot which Royce had suggested as suitable for the camp was a
knoll, on which a few palm-trees grew, at one end of the village, and Challis
was rather surprised to find that, during his conversation with Royce, the
Hausas had not made a start. Wearing woe-begone expressions on their
faces, they were squatting beside their packages.
"Now, Kulana," said Challis to the second in command, "what are you all
idling for? Carry the stuff to those palm-trees yonder."
The men got up obediently, but they appeared to have a strange hesitation
in lifting their loads.
"Come, come!" said Challis. "I know that you are done up, but you are
not so bad as all that. Besides, it's only a few yards away, and the sooner we
form camp the sooner you'll get to sleep."
There was a murmuring among them. Still they did not offer to lift the
bundles.
Challis curbed his rising anger. The men were usually so willing that he
wished not to be hard on them. And both Royce and he had already taken
much interest in studying the queer moods and ideas of these Africans.
"What is troubling them, Kulana?" he asked quietly.
The Hausa looked him full in the face, and, seeing no anger there,
replied:
"No want camp dar, sah—berry bad place."
"Oh! Why is that?"
And then the man began to pour out an explanation of which at first
Challis could make nothing. Kulana's English was not adequate to express
his thoughts. He talked of "debbils" and "bad medicine," and went through a
pantomime of gestures expressing fear and fright, the other men listening
intently, and murmuring approval now and then.
After a time, however, Challis had a glimmering of light.
"You think the spirits of the dead men haunt the village—is that it? And
you are afraid they will come and bother you during the night?"
"Sah savvy all 'bout it," said the man, delighted that his meaning was
understood.
The others clapped their hands, and looked much more cheerful.
"Well, I don't want you to see black ghosts, I'm sure," Challis went on. "I
don't know that I'd like to see them myself. We'll find another place."
The men shouted, and springing to their feet, hoisted their loads with
alacrity. Challis reflected that the work of constructing a camp more in the
open would be good for them, preventing them from brooding over their
troubles, real and imaginary.
After a rapid glance around, he led them to an open spot at some little
distance from the village, and ordered them to make a small zariba with
branches from the bushes around. It was an excellent situation for a camp.
Its openness rendered it possible to detect the approach of an enemy, and a
small stream running close by furnished an ample supply of water.
As he had expected, the work of cutting the bushes took the men's minds
from their misfortunes. They soon began to chatter with their usual
cheerfulness. By nightfall they had constructed a thick fence six feet high
and several yards square. The tent was erected in the middle of the
enclosure, the baggage was placed against one of the walls, and the men,
tired out, but no longer despondent, were ready for their supper.
Challis dealt out pills to the ailing ones, promised them all a long rest on
the following day, and spoke confidently of the speedy return of his
companion with ample supplies. He arranged for the watching of the camp
during the night, and, as an extra precaution, set his alarm clock, when he
himself turned in, so that it wakened him in an hour. Then he took a look
round, set the clock again for an hour ahead, and so on, through the night.
He enjoyed the inestimable power of sleeping at any minute.
The night passed quietly. In the morning, Challis sent two of the men
outside the zariba to fetch water and to take a look round.
When they returned they were rather excited. They explained that they
had made two discoveries. One was that across the stream lay an extensive
swamp, upon which they had seen a large flock of birds pecking for worms.
The other was that a little farther off was a banana plantation, which had
escaped the ravages of the raiders.
"Good boys!" said Challis. "We will make some use of your news by and
by."
The presence of the birds and the bananas afforded a welcome

opportunity of replenishing their scanty larder. Challis decided to go out and
try his skill as a sportsman. There would be some risk, of course, that the
noise of his gun might attract the notice of undesirable visitors; but there was
very little food of any kind left, and poultry would be a treat to the men. In
view of the possibility of Royce's return being delayed, it seemed too good a
chance to be neglected. Besides, he reflected, there was no other village in
the neighbourhood, and the raiders, having accomplished their destructive
work only too completely, were probably by this time far away. After he had
made a bag of birds, he would send out some men to gather bananas, and the
party would revel in a regular banquet.
Accordingly, he ordered Kulana to keep a careful watch, and on no

account to allow the men to move outside the zariba; then, taking his shot-
gun, he set out for the swamp, with the two who had brought the
information, to retrieve the game.
The swamp was half-a-mile away, and Challis found it necessary to walk
round it for some distance before he could get a fair shot at the birds, which
had settled down in a bed of rushes. They appeared to be a variety of pigeon,
a delicacy which the men would appreciate after living almost wholly on
meal.
When he had located the game, he sent his men forward to start them.
The instant they rose in the air he let fly with both barrels in rapid
succession, and brought down a bird with each shot. Following up the rest of
the covey until they settled again, he killed another brace, and so went on for
nearly an hour, until there were as many birds as the men could carry.
His success had quite banished the misgivings with which he had started.
Picturing the delight of his boys at getting a whole day's good eating, he had
given the order to return, and was half-way back to camp, when he was
startled by a great shouting and commotion from its direction.
Sprinting on at full speed, he was just in time to see a number of his boys
running across the open space in front of the zariba, closely pursued by a
dozen horsemen. The runners, who appeared to be laden with bananas,
darted into the enclosure through the gap they had left in the fence; but
before they could draw the rough gate across it, the foremost horsemen
galloped through at their heels.
Challis guessed in a moment what had happened. The men's appetite had
been whetted by the report of the nearness of a banana plantation. They had
been too childishly impatient to await his return, and, either with Kulana's
consent or in defiance of him, they had gone out to gather the fruit, only to
be surprised by some wandering tribe.
He rushed impetuously across the open space to the support of his men,
not staying to reflect that he could do nothing really effective. Besides his
shot-gun, he carried a revolver. The horsemen had fire-arms, which they had
not used as yet, feeling no doubt that their swords were sufficient for the
work in hand. Some of the Hausas had rifles, but it was clear that they were
too much paralysed by the appearance of a mounted enemy to make use of
them. They were yelling with fright.
Before Challis was half-way to the zariba, a second party of horsemen

broke from cover in the direction of the village, and rode straight at him.
Flinging up his shot-gun, he emptied the barrels almost at random; then drew
his revolver. But at that moment a shot from the pistol of one of the
galloping horsemen struck him above the wrist, and the revolver fell to the
ground. In another second he was spun round by the impact of a horse's
shoulder, reeled, and fell. The horsemen galloped over him on their way to
the zariba, and before he lost consciousness it seemed to him that he had
been kicked and trampled by a hundred hoofs.
CHAPTER V
UNDER THE LASH
Challis opened dazed eyes upon a scene that bewildered him, and for a
few moments he could not account for the pains that gripped all his limbs.
Remembrance stole back into his reviving consciousness, and gradually he
became aware of the meaning of what he saw.
The zariba had been demolished. At one side of what had been his camp a
number of horses were tethered. In the centre his Hausa boys were busily
packing the baggage, much more rapidly than Challis had ever seen them
doing it before. The reason of their haste was easily discovered. Over them
stood a circle of negroes, who urged them with fierce cries and drawn
swords. The camp had fallen into the hands of an enemy.
And it was not long before Challis guessed who this enemy was. Only
one tribe in this part of Africa, so far as he knew, rode horses. These men
must be Tubus from across the Yo—the ruthless brigands who were the
terror of the country. It could hardly be doubted that these were the men who
had raided the village, and left only too clear proofs of their merciless
ferocity.
Apparently there had been no fight at the zariba. The Hausas, armed
though they were, had succumbed without a struggle. The truth was that, in
disobedience to Challis's order, some of the men had left the camp, and been
pounced on suddenly by the enemy. As they fled back to the zariba, their
comrades dared not fire for fear of hitting them, and the swift onset of the
horsemen had made resistance hopeless.
Aching all over, Challis struggled to his feet. Immediately a hand was
laid upon him from behind. He noticed that his wrist was bleeding, and
taking out his handkerchief, he began to wind it round the wound as his
captor pushed him towards the centre of the camp. And then from behind the
horses there came forth a huge negro, taller than he by six or seven inches,
with massive shoulders and muscular arms.
The giant's face broke into a grin as he approached the Englishman. He
uttered some words which Challis did not understand, but which seemed to
have in them the ring of triumph.
"I have caught you, white face!" was what he said.
As to the white man all negroes seem at first alike, so to Goruba Challis
at this moment appeared to be the man with whom he had wrestled at dawn
of day.
He spoke again, addressing Challis; then, recognising that he was not

understood, he called for one of the Hausas to come and interpret. Kulana
came up, and keeping his eyes averted from Challis, he translated what the
big man said.
"What are you doing in this country?"
Challis quickly made up his mind to give no information, trusting that he

might at least save Royce from his own fate. He refused to speak. The next
question puzzled him, but confirmed him in his resolution.
"What were you doing in my fort?"
Though he made no reply, it was plain that he showed his surprise in the
expression of his face, for Goruba looked hard at him, and seemed to be in
some puzzlement himself. Then the negro's harsh features darkened with
anger. He flourished his sword.
"Dog! This will make you speak!" he shouted.
Challis looked at him, without quailing, and did not flinch when the
sword was flashed across his eyes. His courage seemed to impress Goruba,
who laughed, spat on the ground, and giving an order to his men to keep
guard over the prisoner, walked away with Kulana into the midst of the
sweating Hausas.
What he had failed to elicit from the Englishman his threats soon
extracted from the carriers. He learnt that there was another white man, who
had set out on the previous afternoon for a village to the north, to buy
provisions. He chuckled on receiving this information. There was little doubt
what village the white man he had met in the fort was bound for. He
chuckled again. The white man was no doubt on his way back to the camp. It
would not be difficult to waylay him.
The work of packing was completed. Goruba ordered the Hausas to

mount their loads. Their rifles, with Challis's, were in the possession of his
men. When all was ready, the Tubus leapt to their saddles, and the whole
party set off northwards, Challis being tied to the saddle of one of the horses.
Often on that march Challis's blood boiled as he saw how his men were
treated, and knew his helplessness to defend them. The Tubus urged them
with whips, sometimes with the points of their swords. The wretched
Hausas, some of whom were weak with sickness, panted along under their
loads, striving to keep pace with the impatient horsemen. They dared not
even groan, for a murmur brought the lash on their shoulders. When Challis
protested through Kulana, explaining that the men were ill, Goruba only
grinned and mocked him.
By-and-by, however, it became apparent to Goruba that the men were

incapable of further marching. The slave-driver is usually callous enough as
to the fate of his victims; he will watch them with unconcern growing
weaker and weaker, see them drop in their tracks, sometimes kill them in
sheer rage at their inability to keep up. But Goruba did not wish to lose these
men. They were themselves valuable. They bore valuable loads. It would be
a mistake to over-drive them. In the afternoon, therefore, some hours earlier
than a march is usually ended, he gave the order to halt. The Hausas laid
down their burdens, and threw themselves on the ground in utter exhaustion.
THE PRISONER
Challis himself was in little better case. He had not been given a load to
carry, but he had felt himself growing weaker and weaker as the day wore
on. Though his wound was not serious, he had lost some blood, and was
enfeebled by the shock and the bruises he had suffered in the trampling.
When he lagged on the march, the man to whose saddle he was fastened
prodded him in the back with the point of his spear. His own sufferings, and
the sufferings of his men, made him realise with new force the horrors of
slave-driving, which, in spite of all efforts to crush it, still exists in parts of
the dark continent.
It was therefore with inexpressible relief that he welcomed the order to

halt. The place chosen for camp was the crest of a slight undulation. The soil
was sandy, and hot from the beating of the sun upon it all day. There were a
few scrubby bushes dotted around, but no grass. Nor was there a stream in
which the marchers could bathe their burning feet.
The Tubus fetched water from a small water-hole near by. They made a
meal of the provisions carried in their wallets. The Hausas consumed the last

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Development in Wastewater Treatment

Research and Processes: Microbial

Development in Wastewater Treatment Research and

Development in Wastewater Treatment Research and

Development in Wastewater Treatment Research and

Advanced Oxidation Processes for Effluent Treatment

Emerging Technologies in Environmental Bioremediation

Catalytic Processes for Water and Wastewater Treatment

Catalytic Processes for Water and Wastewater Treatment

Integrated Environmental Technologies for Wastewater

Copyright © 2022 Elsevier Inc. All rights reserved.

Library of Congress Cataloging-in-Publication Data

For information on all Elsevier publications visit our

Publisher: Susan Dennis

Typeset by TNQ Technologies

CHAPTER 1 Microbial degradation of xenobiotics in bioelectrochemical

2.7.2 Temperature ..................................................................................................33

5.3.2 Recommendations ........................................................................................ 111

7.3.7 Lead ............................................................................................................ 142

9.8 Bioleaching (biomining).......................................................................................186

CHAPTER 11 Microbial-mediated explosives removal and its impact

CHAPTER 13 Counterbalancing common explosive pollutants (TNT, RDX,

14.5.6 Enzymes-based biodegradation of various industrial pollutants ...........324

16.6.1 Nanozymes ...............................................................................................371

18.2.3 Asphaltenes and waxes.............................................................................413

20.3 Oil bioremediation................................................................................................455

Index ................................................................................................................................................. 467

He was well acquainted with the styles of wrestling in vogue in England

The negro's aim seemed to be to throttle his opponent, or to squeeze the

Making a desperate effort to fill his lungs, he uttered a shout, or rather a

"No, sah. Him berry big chap, sure 'nuff."

"Bad fella, sah."

"To make him look pretty, sah."

"Um! They've a queer notion of beauty, then."

Anxious to accomplish his errand and return to Challis, Royce pushed on

"This looks promising," said Royce. "We'll make a spurt, John."

Unconscious of the extravagant claims made on his behalf, Royce leant

"Did you tell him I want to buy food?"

"No, sah; I tell him——"

"Then do so at once," Royce interrupted sternly.

John: "Hail, chief! Is it well with you?"

Chief: "It is well."

John: "Allah be praised! Is it well with your wives?"

Chief: "It is well."

John: "Allah be praised! Is it well with your children?"

Chief: "It is well."

John: "Allah be praised! And your horses?"

Chief: "It is well."

John: "Allah be praised! And your cattle?"

Chief: "They are well."

John: "Allah be praised! Your house and all that is yours?"

Chief: "All is well."

John: "Allah be praised!"

The chief seemed amused at the thought of a wrestling match between an

"What does he mean by Goruba?" asked Royce of John, after a brief

The chief seemed to deliberate whether he should speak or not. At last he

The first thing to do was to form as secure a camp as possible. The

"What is troubling them, Kulana?" he asked quietly.

"No want camp dar, sah—berry bad place."

"Oh! Why is that?"

After a time, however, Challis had a glimmering of light.

The presence of the birds and the bananas afforded a welcome

Accordingly, he ordered Kulana to keep a careful watch, and on no

Before Challis was half-way to the zariba, a second party of horsemen