Maurya Et A (Modern Era of Microbial) 2021

Modern era of microbial
biotechnology: opportunities and 11

future prospects
Deepak Kumar Maurya1, Ashutosh Kumar2, Uma Chaurasiya3,
Touseef Hussain4 and Sanjay K. Singh1
1
Biodiversity and Palaeobiology Group (Fungi), National Fungal Culture Collection of
India, MACS-Agharkar Research Institute, Pune, Maharashtra, India, 2ICAR-Indian
Institute of Seed Science, Mau, Uttar Pradesh, India, 3School of Studies in Botany, Jiwaji
University, Gwalior, Madhya Pradesh, India, 4Plant Pathology Section, Department of
Botany, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
11.1 Introduction
11.1.1 Introduction to microbial biotechnology
Microbial biotechnology is a highly competitive, multidisciplinary subject. Microbial
biotechnology can be broadly defined as the “use of organisms or their products for
commercial purposes.” As such, traditional biotechnology has been practiced since the
beginning of history and it has been used to make/bake bread, brew alcoholic bev-
erages, and breed food crops or domestic animals (Nath et al., 2016). Further, develop-
ments of molecular biology have empowered biological science with prominent tools
that have the potential to manipulate the genetic makeup of organisms. Currently, bio-
technology places more emphasis on the establishment of hybrid genes, followed by
their transfer into organisms in which some of the genes are not usually present (Adrio
and Demain, 2010; Al-Samarai and Al-Kazaz, 2015; Rodrigues and Lira, 2018). In pre-
historic times, a primitive form of biotechnology was practiced by agriculturalists
(Singh, 2019) who established better-quality species of plants and animals by methods
of cross-pollination or cross-breeding (Lammerts van Bueren et al., 2018).
Microbiology is also benefited with acceleration in biotechnology, which
resulted in the development of several beneficial products that have dramatic effects
on the world economy and related society (Kun, 2003; Harzevili and Chen, 2018).
In the modern era, biotechnology and microbiology blended in each other for train-
ing and selective breeding of animals, the cultivation of crops, and the utilization of
microorganisms to produce products such as cheese, yogurt, bread, beer, and wine
(Gupta et al., 2018). However, early microbial biotechnology mostly concentrated
on producing food and industrial applications (Vitorino and Bessa, 2017).
With the advances in classical microbiology, it was discovered that microorgan-
isms, for example, bacteria, yeast, or molds, hydrolyze sugars when they lack
Microbiomes and Plant Health. DOI: https://doi.org/10.1016/B978-0-12-819715-8.00011-2
© 2021 Elsevier Inc. All rights reserved.
318 Microbiomes and Plant Health
oxygen and are ultimately responsible for fermentation (Bokulich and Bamforth,
2013; Nath et al., 2016). This process results in the formation of products (food and
drink). Consequently, fermentation was perhaps first explored by chance, since in
earlier times, nobody knew how it worked. During the prehistoric era, some civili-
zations considered fermentation to be a gift from their Gods. Louis Pasteur first
described the scientific evidence for fermentation in the late 1800s. He demon-
strated a theory known as germ theory, presenting the survival of microorganisms
and their further effects on the process of fermentation. Pasteur’s efforts contributed
to several branches of science.
In the modern era, microbial biotechnology is more complex but highly accepted
(Vitorino and Bessa, 2017). Microbial biotechnology is more effective as a tool in
the development of genetically modified strains and crops with increasing accuracy
concerning specific functions. Areas of microbial biotechnology are spread in dif-
ferent fields. The discovery of new species and their selection, strain improvement
with functional traits have been improving through the standard microbial biotech-
nological applications. Hence, the applications of microbial biotechnology and their
relevance in sustainable development that occurred in a few decades have been
endeavored to cover in the current chapter.
11.1.2 Commencement of microbial biotechnology and allied

sciences
For thousands of years, microorganisms have been used by mankind to supply pro-
ducts such as bread, beer, and wine, but it was limited to human nutrition. In many
articles on the history of biotechnology, it was found that biotechnological techni-
ques were invented in prehistoric civilizations between 5000 and 10,000 BCE.
Egyptian and Indus Valley civilizations used different techniques to conserve and
transform nutrients, baking of bread, and produce alcoholic beverages (Nath et al.,
2016). In 1861 Preysz reported a preservation procedure of wine through heat treat-
ment. After Pasteur’s famous publication in 1865, the Preysz report was not given
legal priority. Enzyme production through the microbial activity was done for the
first-time industrial application in 1894 by Japan scientist Jokichi Takamine in the
United States. Hungarian expert, K. Ereky used the “biotechnology” term in his
book that was published in 1919 (Raju, 2016). The name of classical biotechnology
called a second-phase history of traditional microbial biotechnology due to the
extensive use of fermentation technology for production of various types of organic
acids (citric acid, amino acids, vitamins) and antibiotics by acetone-butanol and
glycerol fermentations (Nath et al., 2016).
The modern biotechnology era was marked in 1950 due to the discovery of the
DNA structure. In this era, DNA sequences were edited with rDNA technology and
monoclonal antibody or hybridoma technology to form new synthetic molecules.
Both technologies made it easy to diagnose parasitology, virology, and cancer. The
sequencing and evolution of human and microbial genomes, the understanding of
the genetic basis of many diseases including the development of antibiotic
Modern era of microbial biotechnology: opportunities and future prospects 319
resistance have led to the development of novel diagnostic procedures, vaccines,

treatments, cures, and management of many diseases (Amarakoon et al., 2017). In
the early 1970s, 40 biopharmaceutical products were produced, such as erythropoie-
tin with the collaboration of traditional industrial microbiology and molecular biol-
ogy. In 1972, recombinant DNA technology was born, which enhanced the
application of biotechnology and gave new light for industrial development (Bhatia
et al., 2015).
A seminar on “New Technologies in Animal Breeding” was sponsored by
European Commission in June 1985. The Animal Breeding Research Organization
(ABRO) and the Poultry Research Centre (PRC) were local organizers of this semi-
nar. Later both merge into the Roslin Institute after 8 years, in 1993, celebrating the
cloning of “Dolly” the sheep. In India, for the development of biotechnology, the
Department of Biotechnology was established by the Ministry of Science and
Technology in 1986 (Raju, 2016). By the end of the 19th century, plant breeding
had been controlled by genetic engineering and advance technologies after
Mendel’s work on the basic principles of heredity revolutionized genetics that led
to the production of transgenic plants in which desirable genes like disease resis-
tance, herbicide resistance, increase shelf-life of fruits could be edited. The crop
was improved by molecular breeding with the use of molecular markers like restric-
tion fragment length polymorphisms and simple sequence repeats that help to pro-
vide a selection of both qualitative and quantitative traits of the crop. In 2002
genetically improved corn and soybeans were cultivated with increased 13% yield
from the year before Indian farmers had obtained insect-resistant cotton from trans-
genic development techniques. Currently, more than 50,000,000 ha of transgenic
plants have been cultivated worldwide. In the last 6 years, more than
170,000,000 ha of transgenic plants have been used worldwide (Bhatia et al., 2015).
Decades later, many new tools have developed from culture-independent methods
for the microbial world. Recently, the whole microbial community is being
explored by metagenomics. Pyrosequencing/next-generation sequencing is also a
useful tool for the detection of stable secondary structure or high GC content
sequencing region (Cardenas and Tiedje, 2008; Siqueira et al., 2012; Kulski, 2016).
11.2 Horizons of microbial biotechnology

11.2.1 Current status of microbial biotechnology, their scope,
techniques, and some examples
The beginning of a modern microbial biotechnology era from the year of 1972, a
new evolution occurred in this field, known as recombinant DNA technology (Khan
et al., 2016). This new technology has boosted new heights and new approaches
established industry in this area. It covers many areas, including recombinant DNA
technology, modern microbial biotechnology, fermentation technology, microbial
physiology, screening for novel metabolites and their strain improvement (Fiedurek
et al., 2017). Some examples are designing of bioreactor and downstream
Figure 11.1 Applications of microbes in microbial biotechnology.
processing, enzyme engineering, cell immobilization, cell fusion, metabolic

engineering, in vitro mutagenesis through modification in protein engineering, and
directed evolution of enzymes to help in applied molecular evolution (Fig. 11.1).
11.2.1.1 Recombinant microorganisms

In microbial genetic discoveries from the 1970s to the 21st century, industrial
microbiology has played a solid structure in revolutionary exploitation and estab-
lishment. Many microorganisms have been used as a host and used for the produc-
tion of recombinant protein, for example, Escherichia coli, Bacillus subtilis,
Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, and Aspergillus
niger (Vieira Gomes et al., 2018). For the successful application and use of recom-
binant microorganisms in higher organisms, such as mammalian and insect cell cul-
ture, and transgenic animals and plants, well-established techniques and experience
are necessary. The progress in microbial biotechnology has been outstanding, and
one of the results which came out is human insulin and human growth hormone
available in the market by genetically engineered bacteria, and it happened within
4 years of the discovery of recombinant DNA technology (Adrio and Demain,
2010; Khan et al., 2016). One part of new technology is that a new company was
established dedicated to innovation via genetic approaches with an exploration of
investment activity and played a role such as biochemical engineering and down-
stream processing. Another interesting one is the production of the vaccine; for
example, hepatitis B, virus surface antigen produced in yeast, the first subunit vac-
cine in the market (Vieira Gomes et al., 2018).
11.2.1.2 Combinatorial biosynthesis

To produce new and altered structures of any product by application of
engineering, to modify the biosynthetic pathways from natural products and all
this was done by using nature’s biosynthetic machinery, known as combinato-
rial biosynthesis (Méndez and Salas, 2001; Sun et al., 2015). Malpartida and
Hopwood (1984) worked on cloning of the biosynthetic genes for the antibiotic
actinorhodin from Streptomyces coelicolor. A clustered gene was responsible
for the microbial biosynthetic pathways. They were facilitating that a single
manipulation, transfer, and change can change the entire pathway. Examples in
fungi, Penicillium and Aspergillus, have penicillin genes and aflatoxin genes,
respectively. These genes are the best examples of pathway genes, which some-
times play role as clustered genes. Recently, recombinant DNA techniques have
been used for the discovery of new or modified secondary products, and to
introduce genes for the synthesis of one product from nonproducing strains is
an example of combinatorial biosynthesis (Zhang and Tang, 2008; Zhang et al.,
2018a).
11.2.1.3 Enzyme production

Before the modern microbial biotechnology era, enzyme production occurred by
fermentation was a conventional business for the industries (Grunwald, 2018).
However, improvement of enzyme production technology in a perfect way by using
recombinant DNA methodology because companies immediately used and involved
in manufacturing enzymes. Recombinant chymosin (cheese manufacture) and
recombinant lipase (detergents) are examples of recombinant DNA technology
(Belenkaya et al., 2018).
11.2.1.4 Biotransformation
Biotransformation, also known as, bioconversion, and it is the conversion of organic
materials, such as plant or animal waste, into usable products or energy sources by
biological processes or agents, such as certain microorganisms (Kaup et al., 2004,
2005; Smitha et al., 2017). Bioconversion is of two types: (1) enzymatic and (2) non-
enzymatic. In enzymatic biotransformation microsomal: enzymes present in the lipo-
philic membranes and in nonmicrosomal: enzyme present in mitochondria are
involved (Donova, 2017). Transformation of steroids and sterols by Corynebacterium
sp. and Nocardia sp. and transformation of nonsteroid compounds such as dihydroxy-
acetone from glycerol by Gluconobacter melanogenus are a few examples (Rokade
et al., 2018).
11.2.1.5 Vitamins
Two yeast-like molds, Eremothecium ashbyii and Ashbya gossypii are reported as
natural overproducer of riboflavin content of vitamin B2. Recombinant B. subtilis
strains or Candida sp. are used for more yield/production of riboflavin by the new
process. Another example of the production of vitamin B12 is by using bacterial

strains of Propionibacterium shermanii or Pseudomonas denitrificans at industrial
scale (Revuelta et al., 2016; Fang et al., 2017).
11.2.1.6 Alcohol
Alcohol (ethyl alcohol) is a fermented product of sugar or a polysaccharide. It is a
primary metabolite of depolymerized fermentable sugar. Hexose sugar is used for
the production of alcohol, the primary substrate, fermented by S. cerevisiae,
whereas lactose or a pentose sugar is substrates fermented by Kluyveromyces fragi-
lis or Candida species, respectively (Shi et al., 2009; Donova, 2017). Recombinant
DNA techniques would be applied/used to make recombinant microbes such as
E. coli, which can convert into an excellent ethanol producer (Khan et al., 2016).
11.2.1.7 Biofuels
Biofuel, reveals that the production of fuel using plants as well as microbes. For
sustainable biofuels production, microbes play an important role in the production
of sustainable biofuels. Currently, two main types of biofuels are commercially pro-
duced, and they are bioethanol and biodiesel. Production of alcohol, generally,
substrate-like sugar (starch) and other plant material have been used for the
fermentation process. Production of biofuels from edible crops is called the first
generation. Microbes play an important role in the development of second and
third-generation biofuel and based on nonfood crops (Antoni et al., 2007; Heo and
Choi, 2019). Biodiesels works as biofuel when alkyl esters of the long fatty acid
chain are used as nonpetroleum-based diesel and produced from animal and plants
(Fukuda et al., 2001; Kumar and Kumar, 2017; Robak and Balcerek, 2018).
Recently, microalgae play a role in the production of third-generation biodiesel
because of their photosynthetic nature and double their biomass within 24 h
(Tollefson, 2008; Robak and Balcerek, 2018). Engineering yeast increases bioetha-
nol production by genetically modifying microorganisms to increase their bioetha-
nol yields as well as biodiesel production (Khan et al., 2017).
11.2.1.8 Biopesticides
Biopesticides are types of chemicals extracted from living organisms such as animals,
plants, and microorganisms, for example, bacteria, fungi, and viruses (Damalas and
Koutroubas, 2018) (Table 11.1).
11.2.1.9 Microbial biotechnology in health care

In 1975 the construction of first hybridomas was announced by Kohler and
Milstein. After several years, this technology not only became widespread, but
appropriate steps of technology have undergone improvement (Tulchinsky and
Varavikova, 2014). To improve human health care, recombinant proteins are gener-
ally used. Thousands of firms all around the world have become a part of the
Table 11.1 Some important commercial available biopesticides.
Biopesticide name Types References

Biofungicides Kasugamycin, polyoxins Law et al. (2017)
Bioinsecticides Nikkomycin, spinosyns Bacci et al.
(2016)
Bioherbicides Bialaphos Marrone (2019)
Antihelminthics Avermectin Marrone (2019)
Ruminant-growth promoters Monensin, lasalocid, salinomycin Ogunade et al.
(2018)
Plant-growth regulators Gibberellins Sharma et al.
(2018)
Immunosuppressants for Cyclosporin A, FK-506, rapamycin Xiao et al.
organ transplants (2018)
Anabolic agents in farm Zearalenone Zhang et al.
animals (2018b)
Uterocontractants Ergot alkaloids Rajasekaran
et al. (2008)
Antitumor agents Doxorubicin, daunorubicin, Mikó et al.
mitomycin, bleomycin (2019)
production technologies of necessary hybridoma technology (Greenwood, 2014).

The role of microorganisms in the field of biotechnology and other applied sections
was in the early 1990s. For example, DNA vaccines have a potential wide-ranging
opportunity to consist of properly engineered plasmid DNA, and it is prepared on a
large scale using E. coli (Singh et al., 2017a).
11.2.1.10 Microorganisms in the treatment of cancer

Among all the human health care issues cancer is a significant challenge in the 21st
century and several populations all over the world are suffering from different types
of cancer. Nowadays, a standard method of treatments is practiced to cure cancer.
Immunotherapy is one of the promising researches in this direction, including the
use of specific types of microbes (Mohammed et al., 2016). The role of microorgan-
isms and their antitumor efficacy is exceptionally diverse. After the results of clini-
cal trials by researchers, a particular product can be projected for general use.
Currently used anticancer bacterial microbial preparations are helping to increase
the chances of complete recovery, and this therapy is complementary to standard
treatment (Łukasiewicz and Fol, 2018) (Table 11.2).
11.2.1.11 Industrial biotechnology

In the long history of biotechnology, fermentation technology is one of the oldest
fields. Many companies are still in the process of synthesis of crude alcohol from
potato, fermentative ethanol from molasses and others such as fermentative vinegar/
baking yeast, and production of drinks, sparkling wines, or beer (Lee et al., 2016).
Table 11.2 List of microorganisms used/planned to be used treatment of cancer.
Microorganism Strain/antigen Cancer

Mycobacterium Attenuates strain Superficial bladder Elzein et al.
bovis Calmette Guérin cancer (2016), Liu
et al. (2019)
Streptococcus OK-432 Lymphangioma Rebuffini et al.
pyogenes (2012)
Clostridium novyi NT and VNP20009 Solid tumors Sedighi et al.
and Salmonella (2019)
enterica
Magnetococcus MC1 Solid tumors and some Łukasiewicz and
marinus metabolic tumors Fol (2018)
Toxoplasma gondii CPS/TLA Pancreas, lung and Sanders et al.
ovarian cancer, and (2016)
melanoma
Plasmodium rVAR2-DT Melanoma expressing Agerbæk et al.
falciparum Chondroitin sulfate (2018)
The use of microorganisms has a very long history of producing fermented foods,
and microbial fermentation is essential for the production of wine, buttermilk,
beer, bologna, sauerkraut, cheese, kefir, salami, olives, and many more (Berger,
2012). The microorganism’s metabolic end products are flavor-fermented foods.
Fungi produce mold-ripened cheeses, ketones, aldehyde mixture, and short-chain
fatty acids (Lee et al., 2016). Another example is the production of peptides and
bacteriocins protein from fermented food by lactic acid bacteria, commonly, and
is widely used for this purpose. The role of bacteriocin protein is to inhibit the
growth of unwanted microbes so that no food spoilage can occur and the multipli-
cation and reproduction of food-borne pathogens are controlled (Okafor and
Okeke, 2017).
11.2.1.12 Agriculturalbiotechnology
The methods of microbial biotechnology significantly increase the diversity of
genes, and they can be incorporated into plants. With the help of this technique, it
can reduce the time required for the production of new varieties of plants.
Transgenic plants are an example of the transfer of genes into the plant cells
(Padikasan et al., 2018). Transgenic plants can grow in unfavorable environments
(Raman, 2017). They can tolerate high moisture and high salt concentrations, along
with extending the habitat range from cold, heat, to drought. Many effective broad-
spectrum herbicides do not distinguish between weeds and crops. Crops plants can
be modified to become resistant to these particular herbicides. When applied to a
weed-infected field of such genetically modified plants, these herbicides act as
selective weed killers (Harding and Raizada, 2015).
11.2.1.13 Plant biotechnology

Nowadays, plants are engineered to produce Bacillus thuringiensis (Bt). B. thurin-
giensis (Bt) is bacterial and can produce a particular type of protein that helps to
kill certain insects, and this bacterium is naturally present in the soil. This protein
disrupts the function of their digestive system after the ingested by the insects
(Castagnola and Jurat-Fuentes, 2012; Abbas, 2018). Another advantage of this bac-
terium is that it is not harmful to animals like humans, fish, birds, and valuable
insects. But, there are several reasons for ineffectiveness of insecticide are photoly-
sis of active compounds by sunlight and run off of insecticide by rain water. So, Bt
is most effective when it is contained in the plant itself (Van Huis et al., 2013;
Abbas, 2018). Xu, in the year 2010, introduced conventional breeding techniques.
They worked on corn to improve each desired trait-like, for example, drought toler-
ance, yield performance, etc. With improved knowledge and technology, agricul-
tural microorganisms will have opportunities to produce advanced and developed,
agricultural products that are environmentally friendly (Wang et al., 2018a,b).
11.2.1.14 Microorganisms in wastewater treatment and

environmental applications
The natural resources of the planet have been using abundantly by humans.
Microorganisms help in the reduction of waste materials that are in the form of
solid or liquid (Dadrasnia et al., 2017). Generally, microbes play an essential role in
the treatment of wastewater and also help in the welfare of life on Earth (Liu et al.,
2018). Bioremediation, biomining, and microbial desulfurization of coal and other
applications can be affected by the manipulation of conditions likes nutrients, oxy-
gen tension, temperature, agitation, etc., which influence the function of the particu-
lar microbial community (Hurst et al., 2007; Dadrasnia et al., 2017).
11.2.1.15 Bioremediation
Bioremediation is the process in which organic wastes are biologically degraded and
depend on the activities of living organisms under controlled conditions to a nontoxic
state or levels below concentration limits (Arora, 2018). In this process, living organisms
(especially microorganisms) are used to degrade the environmental contaminants into
less toxic forms (Atlas and Philp, 2005; Ojuederie and Babalola, 2017). Bacteria and
fungi help to degrade or detoxify substances, which are hazardous to human health and
the surrounding environment. These microbes may be indigenous, or they may be iso-
lated from the contaminated site, and their metabolic process may help to transforms or
enhance the degradation of the contaminated compound. This process is called bioaug-
mentation (Nzila et al., 2016). Bioremediation has its limitations in such pollutants that
have high chlorinated compounds or high aromatic hydrocarbons, as they are resistant to
microbial attack (Igiri et al., 2018). Many factors may be responsible for controlling and
optimization of bioremediation such as population or availability of microbes in pollutant
and environmental factors such as the type of soil, pH, temperature, nutrients, and the
presence of oxygen.
11.2.1.16 Biomining
Biomining is the process by which extraction of heavy metal is done with the help
of microorganisms. Microbial methods are being increasingly applied for leaching
of metal from low-grade ores (copper, nickel, and zinc) and are concentrated espe-
cially ones that cannot be processed economically by conventional methods
(Gumulya et al., 2018; Igiri et al., 2018). Different acidophilic, chemolithoauto-
trophic species utilizes inorganic salts and CO2 as energy sources. For example,
bacteria belonging to the genus Thiobacillus, gram-negative, rod-shaped, aerobic
help partially reduction of sulfur compounds. Gold, copper, and uranium are inert
to microbial action, but at present, biomining is being used for the recovery of gold,
copper, and uranium commercially (Zhou et al., 2018; Donati, 2018).
11.2.1.17 Paper manufacturing industries

Paper manufacturing discharge significant quantities of wastewater and contain an
abundant amount of high molecular weight lignin (a large group of aromatic rigid)
and its derivatives. Certain pure bacterial strains are responsible for the degradation
of 70% 80% lignin (Brown and Chang, 2014). White rot fungi are used to treat
wood, and their extractives have been accountable for cellulose so that it decreases
the toxicity of pulp mill effluent and have a less harmful effect on aquatic organ-
isms (Walia et al., 2017). Wood extractives are obtained during the process of
wood pulping and refining of paper pulp and have compounds such as triglycerides,
esters, resin, sterol, fatty acids, and waxes (Atlas and Philp, 2005; Walia et al.,
2017). Pitch or resin and resin acids are mostly present in softwood and generally
absent or minor components in hardwood species (Walia et al., 2017). Fungi are
usually used for pretreatment of the wood to degrade and can be less toxic to some
of the wood extractives.
11.2.1.18 Microbial biosensors

Last few decades, scientists have taken valuable efforts and success in the develop-
ment of viable biosensors and successfully. It is an analytical device that combines
a biological sensing element with online process monitoring systems (Vigneshvar
et al., 2016). Microbial biosensors have a couple of microorganisms along with a
transducer that enables rapid, sensitive, and accurate detections of target analytes in
the field. The role of the microbial biosensor is diverse, such as food processing,
packaging, safety, environmental monitoring, clinical and defense (Neethirajan
et al., 2018). Recently, genetically modified microbes are being widely used to
manipulate their genes for the detection, assay of toxicity, and bioavailability.
11.2.1.19 Microbiome tools for forensic science

The journey of the microbiome exploration started in the late 19th century in the
field of forensic science. The role of microbes in this field was aimed at the evi-
dence in pathogenicity, and it may also help to find out the cause of the death of
humans and other animals (Metcalf et al., 2017). Earlier, microbiologists such as
Louis Pasteur, Robert Koch, and Joseph Lister worked on it. Half decades of the
20th century played an important role in forensic microbiology due to solving some
cases. In the early 21st century, the development of biotechnology with sequencing
techniques for encoding genetic information resulted from significant advances in
microbial forensics. These techniques help in identifying molecules correctly, and
nowadays, the cost of chemicals and other things is also less expensive.
11.2.1.20 Metagenomics
The modern era of taxonomy has shifted from biochemical to molecular and
focuses on DNA analysis. This process started from individual genes to a complete
set of genes present in an organism and complete on to the genome analysis.
The beginning of the new era of microbial ecology was initiated and intro-
duced to describe uncultured microbes in the environment (Marco, 2010).
Metagenomics is a method that describes the functional and sequence-based
analysis of the collective microbial genomes in an environmental sample. This
diversity of habitats provides a vast number of sites to sample, all with a tremen-
dous potential interest for metagenomic analysis (Breitwieser et al., 2017). The
metagenomic analysis allows determining the presence and relative abundance
of specific genotypes and their interaction with different components of that par-
ticular ecological niche. Metagenomic tools can be handy to expand our under-
standing of microbial diversity and community structure of extreme ecological
habitats (Marco, 2010; Breitwieser et al., 2017) (Fig. 11.2).
Figure 11.2 Metagenomic analysis of environmental microbial communities based on

nucleic acids.
11.2.1.21 Nanobiotechnology
Many microbes have efficacy to produce various nanoparticles (NPs) of metal
oxides such as Fe, Zn, Au, and Ag in nature through either extracellular or intracel-
lular routes (Sanz and Maestro, 2017; Aziz et al., 2015). Some of the common
microbial strains that are capable of synthesis of NPs are Bacillus cereus,
Rhodococcus sp., Candida utilis, E. coli, Pseudomonas aeruginosa, actinomycetes,
Aspergillus flavus, Fusarium oxysporum, Verticillium sp., Trichoderma viride, and
Thermomonospora sp. (Li et al., 2011). The researches of the modern era have
shown immense interest in the formation of NPs from elemental oxides and alloys
due to their wide applications in catalysis, electronics, optical materials, coatings,
and biomedical uses (Li et al., 2011; Lee and Jun, 2019).
11.2.2 Primary metabolites production

11.2.2.1 Primary metabolites
Organic acids and amino acids: Microorganisms can function as resourceful
factories for the production of the primary metabolites at industrial scale and are
the intermediates or end products of the pathways of living cells (Nath et al.,
2016). Metabolites of microorganisms are used in food and feed industries (Nath
et al., 2016). Some organic acids and amino acids come under this category and
are prominent products, for example, different types of amino acids (lysine, threo-
nine, phenylalanine, monosodium glutamate, and tryptophan) (Krämer, 2004;
Nath et al., 2016); organic acids such as citric, succinic, fumaric, lactic, acetic,
and propionic (Burgstaller, 2006; Nath et al., 2016); natural sugars (fructose,
ribose, sorbose); flavor nucleotides (such as 59-inosinic acid and 59-guanylic
acid); essential vitamins [biotin, riboflavin (B2), cyanocobalamin (B12)]; two
types of polysaccharides (xanthan and gellan); and some types of polyols (glyc-
erol, mannitol, erythritol, xylitol) (Nath et al., 2016). Fungi are commonly used in
industries for the production of organic acids, for example, citric acid production.
Embden Meyerhof pathway is used for citric acid production (Schneider et al.,
2014). A. niger is used for the commercial production of citric acid and media
generally deficient in iron and manganese for the production of acids (Bakhiet
and Al-Mokhtar, 2015). In the glycolysis cycle, the concentration of fructose 2,6-
biphosphate acts as an activator and is associated with a high level of citric acid
production. There are alternative processes in which Candida yeasts are generally
used for the production of citric acid by the use of hydrocarbon (Roukas, 2005;
Nath et al., 2016), and they can convert n-paraffins to isocitric acid and citric
acids with high yields. During the major commercial amino acid production like
L-glutamic acid, to increase in outward permeability of microorganisms is a factor
because they are increasing the process of production. Corynebacterium glutami-
cum, Brevibacterium flavum, and Brevibacterium lactofermentum are regularly
used in fermentation technology for the annual production of monosodium gluta-
mate (Kimura, 2003; Eggeling and Sahm, 2003; Adeogun et al., 2017).
11.2.2.2 Glutamic acid

Due to feedback regulation, overproduction of glutamic acid would not occur. Microbial
cell pumps out the glutamate so that some modification may be carried out in the cell
membrane, therefore allowing its biosynthesis to proceed unchanged. All bacteria, which
are producing glutamic acid, are biotin auxotrophs, and limitations in biotin cause an
effect on the membrane alteration (Hirasawa and Shimizu, 2017). So many other com-
pounds or derivatives, which help in exponentially growing cells, for example, the addi-
tion of penicillin or fatty acid derivatives, glycerol limitation of glycerol auxotrophs, and
oleate limitation of oleate auxotrophs (D’Este et al., 2018).
11.2.2.3 Lysine
Lysine is an essential amino acid among all amino acids, and most of the cereals
have a deficiency of L-lysine. Lysine comes under the aspartate family. Lysine is
produced in bacteria by a branched pathway that also produces three other types of
amino acid, for example, methionine, threonine, and isoleucine (Pfefferle et al.,
2003; D’Este et al., 2018). Microorganisms such as E. coli having three aspartate
kinases and mutants of C. glutamicum having single aspartate kinase in lysine regu-
lation control this pathway (Korosh et al., 2017).
11.2.3 Secondary metabolites production

Secondary metabolites are produced only by certain restricted taxonomic groups of
microorganisms by specific pathways and are enormously important for nutrition and
health. Antibiotics, biopesticides, medicinal toxins, and animal and plant growth factors
are a group of metabolites, which are of tremendous economic importance (Baral et al.,
2018). In nature, secondary metabolites are essential for the survival of microorganisms,
and they have no role or function in the growth of the microorganisms (Singh et al.,
2019). These metabolites are closely related members of a chemical family (Baral et al.,
2018).
11.2.3.1 Production and role of antibiotics

Antibiotics are the best example and most important of the secondary metabolites pro-
duced by microorganisms. Among secondary metabolites, there are many different
types or classes of antibiotics discovered (Hug et al., 2018). Some antibiotics such as
actinomycin, bleomycin, and griseofulvin target the DNA replication; rifamycin targets
transcription; chloramphenicol, tetracycline, lincomycin, erythromycin, and streptomy-
cin target translation by 70S ribosomes; cycloheximide targets transcription by targeting
80S ribosomes; puromycin and fusidic acid target transcription by 70- and 80S ribo-
somes; cycloserine, bacitracin, penicillin, cephalosporin, and vancomycin target cell
wall synthesis, respectively (Van Lanen and Shen, 2006). Some other antibiotics such
as polymyxin and amphotericin work as surfactants; monensin works as mobile carrier
ionophores, and linear gramicidin works as a channel forming ionophores in cell mem-
branes (Niu et al., 2016). Nowadays advance research is being carried on a combination
of new antibiotics because of combat of evolving pathogens, naturally resistant bacteria

and fungi, and previously susceptible microbes that have developed resistance (Andrio
and Demain, 2010; Banin et al., 2017). These types of infectious microorganisms have
susceptible and increase their resistance against the existing arsenal of antibiotic drugs
(Fair and Tor, 2014). Some of them show only nominal antimicrobial activity and
sometimes may be altered or modified in their chemical structure to produce new anti-
biotics. This process is known as semisynthesis. These semisynthetic products or new
antibiotics can be used in the therapy of human health and veterinary medicine. These
antibiotics can also be used for farm animals in their growth promotion and plant pro-
tection. Antibiotic production has increased 100 1000-fold from their initial discovery
to the present time. There has been a tremendous increase in fermentation technology
and the productivity of compounds and the resulting decrease in costs due to random
mutagenesis and screening for higher-producing microbial strains.
11.2.3.2 Useful nonantibiotic agents

Microorganisms culture filtrates have been screened based on antimicrobial activity,
but also for the presence of other activities such as particular animal enzymes inhibition
or binding to specific receptors. On this basis, several compounds were discovered,
which have this activity, and they do not fit according to the traditional definition of
antibiotics. In nature, secondary metabolites are produced by microorganisms; they are
important and effective, such as sex hormones; ionophores; competitive weapons
against other microbes. They also play an important role against amoebae, insects, and
plants; agents of symbiosis; and effectors of differentiation, respectively. They are used
for noninfectious or noncommunicable diseases from several years by most pharmaceu-
tical companies (Demain, 1996; Pereira et al., 2018). Only a few promising structures
of the compound have found after the testing of thousands of synthetic compounds,
and it is time-taking process. Some of the lead synthetic compounds became most diffi-
cult to find. For example, monensin, lasalocid, and salinomycin are polyethers, and
they are a microbial product. Antitumor agents such as doxorubicin and daunorubicin
were isolated from microorganisms belonging to the anthracycline family. The aver-
mectins are highly effective against arthropods and helminths. Avermectins come under
the other group of Streptomycetes. Using simple enzymatic assay techniques uses in
drug identification, screening of inhibitor and their important activity in pharmacologi-
cal studies (Demain, 1998; Allen et al., 2014). Massive success came in this field after
the two fungal products such as lovastatin is a statin drug used as cholesterol-lowering
agents in humans and pravastatin in animals, respectively (Table 11.3).
11.3 Microbial biotechnology in agriculture

11.3.1 Microbial biotechnology in sustainable agriculture
Under continuous changing climatic conditions, the top priority concern in the cur-
rent scenario is to elevate agriculture production for the growing human population
Table 11.3 Difference between primary versus secondary metabolites (Thirumurugan

et al., 2018).
Primary metabolites Secondary metabolites

Perform physiological functions Derivatives of primary metabolites
Directly involved: growth, development, Play a role in the ecological function
and reproduction
Emerged during the growth phase due to Developed at the stationary phase of
the energy metabolism growth
Produced in large quantities and extraction Produced in small quantities and extraction
is easy is difficult
The basic structural molecule of an Not a basic molecular structure of an
organism organism
Examples are ethanol, citric acid, lactic Examples are antibiotics, pigments,
acid, nucleic acid, proteins, and vitamins essential oil, peptides, terpenoids, and
drugs
in a sustainable way. Agriculture challenges such as soil fertility, productivity

improvement, minimization of synthetic fertilizers, reimbursement of agriculture
land, and incorporation of new technology for the development of a sustainable
agriculture system are few constraints in gaining higher crop production. Further,
prolonged and injudicious application of synthetic fertilizers and pesticides have an
outcome in the degradation of soil health and raise the issue of the agriculture sus-
tainability (Pingali, 2012; Singh et al., 2017b). Land deterioration by application of
pesticides and inorganic fertilizers leads to a change in microbial diversity (Kumar
et al., 2018). The current agricultural practices no doubt enhances crop production,
but at the same time, long-term uses of chemicals will make the soil unfit for agri-
culture in the future. Hence, the uses of microbial technology have been focused
and mentioned as the most appropriate appraiser for the maintenance of soil sustain-
ability without disturbing the soil ecology or climatic variables. Further, uses of
microbial-based products and microbes also have the potential to limit the use of
chemically synthetic in agronomic practices (Singh et al., 2010,2017c; Sengupta
and Gunri, 2015; Schäfer and Adams, 2015). Microorganisms interact in rhizo-
sphere and benefit to standing crop in various ways such as nutrient recycling, envi-
ronmental detoxification, upgrading soil health through different mechanisms such
as nitrogen fixation, accessibility of required plant nutrients (P, K), bioleaching of
inorganic heavy metals, inhibition of plant pathogens, bioprocessing of hazardous
toxicants of pesticides, and secretion of bioactive secondary metabolites (Santos
et al., 2012; Sengupta and Gunri, 2015; Singh et al., 2017a).
11.3.1.1 Role of bacteria, Cyanobacteria, and fungi in agriculture

Higher crop productivity demands an intensive amount of synthetic fertilizers and
pesticides. This prolongs agricultural practice with the application of fertilizers and
pesticides have negatively affected the soil through the reduction of the rhizospheric
microbial population. Hence, the use of microorganisms in the form of biofertilizers

having plant growth-promoting activities (plant growth-promoting rhizobacteria,
PGPR) has been considered an alternative option for soil quality improvement. The
biofertilizers possess several beneficial distinctiveness for enhancing crop produc-
tivity and cut off the high agriculture input (Mahanty et al., 2017). Broadly, the bio-
fertilizers can be classified based on their functions as nitrogen-fixing (free-living,
symbiotic, associative symbiotic), phosphate solubilizing (Arbuscular mycorrhiza,
Ectomycorrhiza, Ericoid mycorrhizae, Orchid mycorrhiza) and PGPR. Some of the
microorganisms commercially exploited for the manufacturing of biofertilizers.
Some of the putative roles of microbes in agriculture sustainability are covered in
Table 11.4.
Besides the growth-promoting activities performed by microorganisms, several
microbes have potential in inhibition of plant pathogens growth through various
mechanisms and regarded as biopesticides. Biopesticides have been engaged in agricul-
tural practices to eradicate insects, diseases, weeds, and nematodes from the standing
crops (Gupta and Dikshit, 2010; Dutta, 2015). Biopesticides of microbial origin provide
protection from a diverse group of pests, and each type of active compound derived
from a single microbe can have a multifunctional spectrum against plant pathogens.
Bacterial and fungal pesticides protect against pests such as beetle larvae, flies,
caterpillars, fungal, soil-borne microbial pathogens, and bacterial diseases. Generally,
fungi assist in inhibiting/limiting sucking insect pests. Some of the widely accepted
species such as B. thuringiensis, Pseudomonas spp., Metarhizium anisopliae, Beauveria
bassiana, Paecilomyces farinosus, Nomuraea rileyi, and Verticillium lecanii work
against pests such as nematodes, aphids, grasshoppers, mealybugs, whiteflies, and soil-
borne pathogens.
Microbial pesticides have been applied against viruses such as cytoplasmic poly-
hedrosis viruses, granuloviruses (GVs), nucleopolyhedrosis viruses (NPVs), DNA-
containing baculoviruses (BVs), and the RNA-containing reoviruses, nodaviruses,
retroviruses, and picrona-like viruses. Further, the main categories applied in pest
management have been included from GVs and NPVs. The biopesticides viruses
are commonly applied for inhibiting vegetable pests, and equally, show capability
against plant chewing insects. Virus microbial pesticides have substantial effective-
ness against different forest pests such as Douglas fir tussock moths, pine sawflies,
gypsy moths, and pine caterpillars. Lacey et al. (2008) reported Cydia pomonella,
effective in controlling the Codling moth in a fruit tree. Arthurs et al. (2008) had
concluded that Phthorimaea operculella is effective in controlling potato tuber
worm in stored condition tubers. Virus-based products are widely applied to control
the cabbage moths, tobacco budworms, corn earworms, cotton leaf worms, and cel-
ery loppers bollworms.
Protozoa and yeast pesticides are also exploited against pests such as grasshop-
pers, crickets, caterpillars, locusts, leaf spot, greasy spot, and fruit drop. Mostly,
nematodes are entomopathogenic and used for controlling the growth of weevils,
white grubs, and several species of the Sesiidae family including Steinernema car-
pocapsae, Seinernema riobrave, Seinernema glaseri, Heterorhabditis bacterio-
phora, and Heterorhabditis megidis (Shapiro-Ilan et al., 2006).
Table 11.4 Role of bacteria, Cyanobacteria, and fungi in agriculture.
Beneficial microbes Functions References
Bacteria
Bacillus spp. Biological control Wang et al. (2018a,b)
Pseudomonas spp. Plant growth Kumar et al. (2017)
promotion and
disease
management
Saccharomyces spp. Zinc solubilizing Raj (2007)
biofertilizers
Bradyrhizobium spp. and Streptomyces Plant growth, Htwe et al. (2019)
griseoflavus nodulation,
nitrogen fixation
Burkholderia spp./Nitrosomonas spp. Nitrogen fixation in Draghi et al. (2018)
legume nodules
Fungi
Trichoderma spp. (Trichoderma Biofertilizer Harman et al. (2004),
harzianum/Trichoderma virens/ enriching compost Singh and Singh
Trichoderma asperellum) (2008)
Beauveria bassiana/Metarhizium spp./ microbial insecticide Mascarin and
Nomuraea rileyi/Verticillium lecanii Jaronski (2016),
Patil et al. (2014)
Paecilomyces lilacinus Nematode Hano and Khan
(2016
Aspergillus spp. (Aspergillus Phosphorous Akintokun
tubingensis/Aspergillus niger/ solubilizing et al. (2007),
Aspergillus terreus/Aspergillus biofertilizers Bhattacharya et al.
awamori/Aspergillus fumigatus/ (2015)
Aspergillus melleus)/Trichoderma
spp. (Trichoderma viridi/T.
harzianum/T. virens/T. asperellum)
Aspergillus spp. (A. fumigates/ Potash solubilizing Lian et al. (2008)
A. niger/A. terreus) biofertilizers
Cyanobacteria/algae
Azolla, Gunner Fix atmospheric N2 Yadav et al. (2014)
Anabaena, Anabaenopsis, Calothrix, Plant growth Singh et al. (2016)
Nostoc promotion
Anabaena spp./Nostoc spp. Phosphate uptake Singh et al. (2014)
11.3.2 Biochemical pesticides

Biochemical pesticides are extracts or secondary metabolites derived from plants, ani-
mals, or microbes that are efficiently applied in controlling pests include substances
such as pheromones, leave extracts, juices, and latex. Dhanasekaran and Thangaraj
(2014) reported that approximately 1500 naturally occurring microorganisms had been
identified as insecticidal agents. Primary and secondary metabolites products from 942
microbial isolates were characterized for their insecticidal and inhibitory properties.
The microbial strains that have inhibitory properties include Streptomycetes and
Actinobacteria that present 18 identified genera and 28 unidentified aerobic genera.
Further, around 70 fungi and 40 bacteria also exhibited similar repulsive odor to insect
and hence are an active mode for checking crop pest’s population. Biochemical pesti-
cides interfere with the mating or breeding cycle of different insects and thereby help
in controlling their population (Gupta and Dikshit, 2010; Dutta, 2015). Biopesticides
such as B. thuringiensis, NPV, and neem-based pesticides are in trend as commercial
sources of biopesticides. Due to the specificity in microbial action, microbes can con-
trol a small portion of the pests infested in a field. The microbial biopesticides have
limitations such as heat, ultraviolet light and desiccation condition on field. Other hin-
drances to microbial-based biopesticides failure are delivery systems, unique formula-
tions, storage procedures, and shelf life.
11.3.3 Genetically modified organisms and transgenic crops

Genetically modified organisms (GMOs) are the plants, animals, or microbes modi-
fied through in vivo techniques to gain specific traits or improved nutritional content.
The modification process involves the transfer protocols of desired or specific gene
(s) from the chromosome of a particular plant, animal, or any other microorganisms
into the host cell species (Gasson and Burke, 2001; Arya, 2015). GMO comes with
developed insect-resistant plants (B. thuringiensis), herbicide resistance, disease resis-
tance, nutritionally enriched crop (golden rice), and other benefits (Dona and
Arvanitoyannis, 2009; Zilberman et al., 2018). Some of the common GMOs are soy-
bean, corn cotton and tomato, brinjal, rice, and many others have been genetically
manipulated to enhance either higher yield or size and durability (Mishra and Singh,
2013; Singh et al., 2017b; Potrykus, 2017; Kumar et al., 2019). Genetically manipu-
lated organisms also cover microorganisms (bacteria and yeast) that have specialized
activity than wild strains (Mishra and Singh, 2013; Zimny et al., 2019).
11.4 Conclusion
Several industrial products such as ethanol, organic acids, amino acids, and others
are produced through the application of microbial technology. The discovery of
new species and their selection, strain improvement with unique functions, the stan-
dard microbial technology and drug manufacturing, microbial-based biosensors
development, GMOs are an extension of microbial technology. Simultaneously,
microbial technology has played an essential role in agriculture production and its
sustainability. In the modern era, microbial biotechnology is more complex and
applied; however, the integration of microbial biotechnology and cutting-edge
research with other basic science will lead to more advances in the field of strain
improvement. Improved microbial strains had potential to manipulate the issues
like xenobiotic degradation, enhance agriculture yield, rapid drug manufacturing,

and primary as well as secondary metabolites production. The development of more
efficient biosensors can be used in the monitoring and maintenance of various
anthropological activities (environmental pollutions).
Acknowledgements
Authors extend heartiest thanks to Directors, ICAR-IISS, Mau, MACS’ Agharkar Research
Institute, Pune and Authority of AMU and University Grant Commission, New Delhi for pro-
viding facilities and working platform.
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Modern era of microbial

biotechnology: opportunities and 11

11.1.2 Commencement of microbial biotechnology and allied

resistance have led to the development of novel diagnostic procedures, vaccines,

11.2 Horizons of microbial biotechnology

Figure 11.1 Applications of microbes in microbial biotechnology.

processing, enzyme engineering, cell immobilization, cell fusion, metabolic

11.2.1.1 Recombinant microorganisms

11.2.1.2 Combinatorial biosynthesis

11.2.1.3 Enzyme production

process. Another example of the production of vitamin B12 is by using bacterial

11.2.1.9 Microbial biotechnology in health care

Table 11.1 Some important commercial available biopesticides.

Biopesticide name Types References

production technologies of necessary hybridoma technology (Greenwood, 2014).

11.2.1.10 Microorganisms in the treatment of cancer

11.2.1.11 Industrial biotechnology

Table 11.2 List of microorganisms used/planned to be used treatment of cancer.

Microorganism Strain/antigen Cancer

11.2.1.13 Plant biotechnology

11.2.1.14 Microorganisms in wastewater treatment and

11.2.1.17 Paper manufacturing industries

11.2.1.18 Microbial biosensors

11.2.1.19 Microbiome tools for forensic science

Figure 11.2 Metagenomic analysis of environmental microbial communities based on

11.2.2 Primary metabolites production

11.2.2.2 Glutamic acid

11.2.3 Secondary metabolites production

11.2.3.1 Production and role of antibiotics

of new antibiotics because of combat of evolving pathogens, naturally resistant bacteria

11.2.3.2 Useful nonantibiotic agents

11.3 Microbial biotechnology in agriculture

Table 11.3 Difference between primary versus secondary metabolites (Thirumurugan

Primary metabolites Secondary metabolites

in a sustainable way. Agriculture challenges such as soil fertility, productivity

11.3.1.1 Role of bacteria, Cyanobacteria, and fungi in agriculture

microbial population. Hence, the use of microorganisms in the form of biofertilizers

Table 11.4 Role of bacteria, Cyanobacteria, and fungi in agriculture.

Beneficial microbes Functions References

11.3.2 Biochemical pesticides

11.3.3 Genetically modified organisms and transgenic crops

like xenobiotic degradation, enhance agriculture yield, rapid drug manufacturing,

Xu, Y., 2010. Molecular Plant Breeding. CABI, ISBN 9781845933920.

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