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This book focuses on successful application of microbial biotechnology in areas such as medicine, agriculture, environment and human health.



Chapter 1. Microbial Applications in Agriculture and the Environment: A Broad Perspective

Microbial diversity is an important component of the overall global biological diversity. Recent technological advances in exploring microbial diversity have revealed that a large proportion of microorganisms are still undiscovered, and their ecological roles are largely unknown. Careful selection of microbes and intelligent design of test assays are the key steps in developing new technologies for effective utilization of microorganisms for sustainable agriculture, environmental protection, and human and animal health. Several microbial applications are widely known in solving major agricultural (i.e., crop productivity, plant health protection, and soil health maintenance) and environmental issues (i.e., bioremediation of soil and water from organic and inorganic pollutants). Wastewater treatment and recycling of agricultural and industrial wastes are other important uses of microbial technology. It is expected that microbes in combination with developments in electronics, software, digital imaging, and nanotechnology will play a significant role in solving global problems of the twenty-first century, including climate change. These advances are expected to enhance sustainability of agriculture and the environment. This chapter provides an overview of recent trends in microbial exploitation in plant growth promotion and sustainable environment mainly through bioremediation, biodegradation, and biosorption processes. Recent uses and application of microbes such as biosensors, synthesis of nanomaterials, and probiotics are also discussed.
Iqbal Ahmad, Mohd Sajjad Ahmad Khan, Farrukh Aqil, Mahipal Singh

Chapter 2. Molecular Techniques to Assess Microbial Community Structure, Function, and Dynamics in the Environment

Culture-based methods are important in investigating the microbial ecology of natural and anthropogenically impacted environments, but they are extremely biased in their evaluation of microbial genetic diversity by selecting a particular population of microorganisms. With recent advances in genomics and sequencing technologies, microbial community analyses using culture-independent molecular techniques have initiated a new era of microbial ecology. Molecular analyses of environmental communities have revealed that the cultivable fraction represents <1% of the total number of prokaryotic species present in any given sample. A variety of molecular methods based on direct isolation and analysis of nucleic acids, proteins, and lipids from environmental samples have been discovered and revealed structural and functional information about microbial communities. Molecular approaches such as genetic fingerprinting, metagenomics, metaproteomics, metatranscriptomics, and proteogenomics are vital for discovering and characterizing the vast microbial diversity and understanding their interactions with biotic and abiotic environmental factors. This chapter summarizes recent progress in the area of molecular microbial ecology with an emphasis on novel techniques and approaches that offer new insights into the phylogenetic and functional diversity of microbial assemblages. The advantages and pitfalls of commonly used molecular methods to investigate microbial communities are discussed. The potential applications of each molecular technique and how they can be combined for a greater comprehensive assessment of microbial diversity has been illustrated with example studies.
Gurdeep Rastogi, Rajesh K. Sani

Chapter 3. The Biofilm Returns: Microbial Life at the Interface

Scientific studies over the past few decades have shown that the vast majority of microbes in the aqueous environment do not live as free-floating (i.e., planktonic) forms, but rather prefer to live as attached communities termed biofilms. Biofilm formation onto surfaces is usually detrimental to human health and man-made structures; biofilm-related problems range from antibiotic-resistant infections in humans and animals to drinking water contamination, energy loss in industrial systems, and increased corrosion in ship hulls and offshore structures. Biofilms also play several beneficial roles, such as nutrient transformation in the plant rhizosphere and enhanced biodegradation of organic carbon and various pollutants during wastewater treatment and soil bioremediation. Recently, biofilms have shown great potential in selective, low-cost catalysis and energy conversion processes in biofuel production and microbially driven batteries. The biofilm structure provides several advantages to microorganisms within the biofilm, including resistance to biocides and antibiotics, viscoelasticity, and resistance against fluid-dynamic shear stress. The congregation of multiple species into biofilm microcosms increases the range of substrates that can be biodegraded and offers great flexibility for a number of biotechnological applications. In the last 20 years, researchers have unveiled the relationship between biofilm structure and activity and have devised many methods to control biofilm development. However, use of biofilms for contaminant degradation in the field is still in its infancy. Furthermore, the processes that employ biofilms for energy conversion, environmental sensing, and “white biotechnology” (commonly known as industrial biotechnology) are still largely confined to academic research. In this chapter, we aim to highlight the most important and recent advances in the field of biofilm-based technologies and their potential applications.
Anand Jain, Enrico Marsili, Narayan B. Bhosle

Chapter 4. Future Application of Probiotics: A Boon from Dairy Biology

Microbes have been used for millenia in food and alcoholic ­fermentations; in recent years, microbes have undergone scientific scrutiny of their ability for preventive and therapeutic effects in humans. This work has led to the establishment of a new term, “probiotics.” Lactic acid bacteria (LAB) are normal microflora of the intestine of most animals. They play an important role in humans and other animals and act as an immunomodulator. LAB are helpful in disease treatment and prevention, as well as for improved digestion and absorption of nutrients. Probiotic microorganisms include LAB i.e., Lactobacillus acidophilus, L. bulgaricus, L. casei, L. plantarum, L. rhamnosus, etc. Use of these live bacteria to elicit an immune response or to carry a vaccine component is a new development in vaccine formulation. The advantages of live bacterial vaccines are their ability to mimic the natural infection, their intrinsic adjuvant properties, and that they can be administered orally. Components of pathogenic and nonpathogenic food-related microbes are currently being evaluated as candidates for oral vaccines.
Vinod Singh, Sarika Amdekar, Hariom Yadav, Nagendra Nath Mishra, Shailini Jain

Chapter 5. Microbially Synthesized Nanoparticles: Scope and Applications

The critical need for development of reliable and eco-friendly processes for synthesis of metallic nanoparticles has recently been realized in the field of nanotechnology. Increasing awareness toward green chemistry and biological ­processes has elicited a desire to explore environmentally friendly approaches for the synthesis of nanoparticles as a safer alternative to physical and chemical methods, which involves harsh conditions and use of hazardous chemicals. Therefore, the use of natural resources, including bacteria and fungi, has been exploited for cost-­effective and environmentally nonhazardous nanoparticle synthesis. The rich microbial diversity of bacteria and fungi contains the innate potential for the synthesis of nanoparticles and may be regarded as potential biofactories. In fact, microbial ­synthesis of nanoparticles has emerged as an important branch of nanobiotechnology. The synthesis of inorganic materials by biological systems occurs through remarkable processes at ambient temperature and pressures and neutral pH. Among the various biological systems, bacteria are relatively easy to manipulate genetically, whereas fungi have an advantage of easy handling during downstream processing and large-scale production. In spite of the successes achieved in biological synthesis of nanoparticles, there is still a need to improve the rate of synthesis and monodispersity of nanoparticles. Also, microbial cultivation and downstream processing techniques must be improved, and more efficient methods should be developed. Furthermore, in order to exploit the system to its maximum potential, it is essential to understand the biochemical and molecular mechanisms involved in nanoparticle synthesis. Delineation of specific genomic pathways and characterization of gene products involved in biosynthesis of nanoparticles are required. The underlying molecular mechanisms that mediate microbial synthesis of nanoparticles will help in understanding the molecular switches and factors necessary to control the size and shape, as well as crystallinity of nanoparticles. Indeed, biological systems are still relatively unexplored, and therefore, the opportunities are open for budding nanobiotechnologists to utilize nonpathogenic biological systems for metallic nanoparticle synthesis with commercial perspectives.
Javed Musarrat, Sourabh Dwivedi, Braj Raj Singh, Quaiser Saquib, Abdulaziz A. Al-Khedhairy

Chapter 6. Bacterial Quorum Sensing and Its Interference: Methods and Significance

Bacteria use the language of low-molecular-weight ligands to assess their population densities in a process called quorum sensing (QS). Different types of quorum sensing pathways are present in Gram-negative and Gram-positive bacteria. Signal molecules most commonly used in Gram-negative bacteria are acyl homoserine lactones. In recent years, a substantial amount of literature and data have been available on bacterial QS. Recently, interest in modulation of quorum sensing with different approaches has increased among scientific communities. In this chapter, we provide an updated overview on bacterial QS, assays and methods for detecting signal molecules, and various approaches to inhibit AHL-based quorum sensing. Significance of QS interference by prokaryotic and eukaryotic organisms in relation to plant health and the environment is discussed here.
Iqbal Ahmad, Mohd Sajjad Ahmad Khan, Fohad Mabood Husain, Maryam Zahin, Mahipal Singh

Chapter 7. Horizontal Gene Transfer Between Bacteria Under Natural Conditions

Conjugative plasmid transfer is the most important mechanism for bacteria to deliver and acquire genetic information to cope with rapidly changing environmental conditions. An update of knowledge of conjugative plasmid transfer in aquatic and terrestrial habitats, including environments of particular concern such as agricultural areas and contaminated soils and sediments, is presented. Environmental factors affecting horizontal gene transfer in nature are discussed. Recent advances in the design of in situ monitoring tools to assess conjugative plasmid transfer in nature and laboratory model systems to simulate environmental conditions are critically reviewed. The impacts of horizontal gene transfer on biodegradation as well as recent approaches to model conjugative plasmid transfer in complex microbial communities are presented.
Elisabeth Grohmann

Chapter 8. Molecular Strategies: Detection of Foodborne Bacterial Pathogens

Conventional methods of pathogen identification have often depended on the identification of disease symptoms, isolation, and culturing of the organisms, and identification by morphology and biochemical tests. The major limitations of these culture-based morphological approaches, however, are the reliance on the ability of the organism to be cultured, the time-consuming nature, and requirement of extensive taxonomic expertise. The use of molecular methods can circumvent many of these shortcomings. Accordingly, there have been significant developments in the area of molecular detection of bacterial pathogens in the last 3 decades. We report here a brief overview of the molecular detection methods applicable to microbes from food.
Javed Ahamad Khan, R. S. Rathore, Iqbal Ahmad, Shaheen Khan

Chapter 9. Recent Advances in Bioremediation of Contaminated Soil and Water Using Microbial Surfactants

Environmental contamination by improper disposal of industrial, ­mining, agricultural, municipal, and other residues is known worldwide. Various ­chemical-, physical-, and biological-based methods are currently being developed for removal of such pollutants from soil and water. Among these techniques, biological ­treatment, or remediation using microbes, is one of the most promising techniques, mainly because of its cost-effectiveness and essentially complete destruction of numerous pollutants. The major requirement for this technique is survivability of the degrading microorganisms during the process. Biosurfactants, particularly microbial surfactants, play a vital role in cases where pollutants are not readily bioavailable, by increasing the apparent water solubility of the pollutants, which could be achieved either by ex situ addition or in situ production of biosurfactants by microbes. However, due to wide application potential of microbial surfactants in the environmental sector, it is important to know their mechanisms of action, recent advances in bioremediation processes, and other possible applications. The goal of this chapter is, therefore, to provide an overview of the different types of microbial surfactants and sources, their roles in several bioremediation processes, and recent advances in the field.
Achlesh Daverey, Kannan Pakshirajan

Chapter 10. Bioaugmentation-Assisted Phytoextraction Applied to Metal-Contaminated Soils: State of the Art and Future Prospects

Bioaugmentation-assisted phytoextraction is a promising method for accelerating the cleanup rate of soils contaminated by metals. On average, bioaugmentation increases metal accumulated by plant shoots by factors of about two (metal concentration) and five, as a result of higher bioaccessibility of metals in soils, with few obvious differences between effects by bacteria or fungi (e.g., plant growth-promoting rhizobacteria and arbuscular mycorrhizal fungi). Metal bioaccessibility is always controlled by microbial siderophores as well as organic acids and surfactants. In cases of excess concentrations, fungi immobilize metals, in contrast to bacteria. Unfortunately, the typically low inoculant survival rate may impair bioaugmentation efficiency. In this chapter, microbial inoculant formulations and management are addressed, as well as strategies for selecting the most relevant plant–microorganism couples for optimum phytoextraction of soil metals. In environments subject to variable conditions, ecological engineering approaches may help in attaining maximal efficiency. Experiments at field-scale are reported, and environmental effects of the technique are discussed. Finally, future prospects are addressed with the main question being how maximal concentrations and amounts of metals in plants can be attained.
Thierry Lebeau, Karine Jézéquel, Armelle Braud

Chapter 11. Biosorption of Uranium for Environmental Applications Using Bacteria Isolated from the Uranium Deposits

Attempts were made to recover uranium (  U  ) occurring in nuclear fuel effluents and mine tailings using bacteria isolated from U deposits in Canada, the United States, Australia, and Japan. To establish which microorganisms accumulate the most U, hundreds of strains of microorganisms were screened. Extremely high U accumulating ability was detected in some bacteria isolated from North American U deposits. Arthrobacter and Bacillus sp. accumulated approx. 2,500 μmol U/g dry wt. of microbial cells within 1 h. Cells removed U from refining wastewater with high efficiency. Cells also accumulated thorium with high efficiency. Lactobacillus cells isolated from Japanese U deposits removed more U from seawater than the other bacteria that had superior U removal capacity from nonsaline U solutions. Cells immobilized with polyacrylamide gel had excellent handling characteristics and can be used repeatedly in U adsorption–desorption cycles. These bacteria from U deposits can be used as an adsorbing agent for the removal of the nuclear fuel elements, which may be present in nuclear effluents, mine tailings, seawater, and other waste sources.
Takehiko Tsuruta

Chapter 12. Bacterial Biosorption: A Technique for Remediation of Heavy Metals

Bacterial biosorption can be used for the removal of pollutants from waters contaminated with pollutants that are not easily biodegradable, such as metals and dyes. A variety of biomaterials are known to bind these pollutants including bacteria, fungi, algae, and certain industrial and agricultural wastes. Biosorbents are less costly and more effective alternatives for the removal of metallic elements, especially heavy metals, from aqueous solution. In this chapter, the sorption abilities of bacterial biomass toward metal ions are emphasized. The appropriate conditions for immobilizing bacteria for maximum biosorption and the mechanism(s) involved are highlighted. The properties of cell wall constituents, such as peptidoglycan, and the role of functional groups, such as carboxyl, amine, and phosphonate, are discussed on the basis of their biosorption potentials. Binding mechanisms as well as the parameters influencing passive uptake of pollutants are analyzed. A detailed description of isotherm and kinetic models and the importance of mechanistic modeling are presented. To enhance biosorption capacity, biomass modifications through chemical methods and genetic engineering are needed for the effective removal of metal. For continuous treatment of effluents, a packed column configuration is suggested and the factors influencing its performance are discussed. The chapter also highlights the necessity for examination of biosorbents within real-world situations, as competition between solutes and water quality may affect biosorption performance. Thus, this chapter reviews the achievements and current status of biosorption technology and provides insights into this research frontier.
Mohd Ikram Ansari, Farhana Masood, Abdul Malik

Chapter 13. Metal Tolerance and Biosorption Potential of Soil Fungi: Applications for a Green and Clean Water Treatment Technology

Heavy metals pose a significant ecological and public health hazard because of their toxic effects and their ability to accumulate in terrestrial and aquatic food chains. This chapter addresses the interactions of heavy metals with organisms for application in wastewater or soil treatment systems, with special emphasis on yeasts and fungi. Conventional techniques to remove metals from wastewaters have several disadvantages; however, biosorption has demonstrated significant metal removal performance from large volumes of effluents. One key step of treatment processes for cleanup of heavy metal-enriched water or soil involves growing resistant cells that accumulate metals to optimize removal through a combination of biosorption and continuous metabolic uptake. Fungal biosorption can be used for the removal of metals from contaminated water and soil; fungal biosorbents are less expensive and more effective alternatives for the removal of metallic elements, especially heavy metals, from aqueous solution. In this chapter, the biosorption abilities of fungal biomass toward metal ions are emphasized. The chapter also highlights the mechanisms involved in fungal biosorption and the factors affecting the biosorption process. The current status and achievements of fungal biosorption technologies are reviewed.
Iqbal Ahmad, Mohd Imran, Mohd Ikram Ansari, Abdul Malik, John Pichtel

Chapter 14. Rhizosphere and Root Colonization by Bacterial Inoculants and Their Monitoring Methods: A Critical Area in PGPR Research

Roots serve a multitude of functions in plants including anchorage, acquisition of nutrients and water, and production of exudates with growth regulatory properties. The root–soil interface, or rhizosphere, is the site of greatest biological and chemical activity within the soil matrix. Plant growth-promoting rhizobacteria (PGPR) are known to influence plant health by controlling plant pathogens or via direct enhancement of plant development in the laboratory and in greenhouse experiments. Unfortunately, however, results in the field have been less consistent. The colonization of roots by inoculated bacteria is an important step in the interaction between beneficial bacteria and the host plant. However, colonization is a complex phenomenon influenced by many biotic and abiotic parameters, some of which are only now apparent. Monitoring fate and metabolic activity of microbial inoculants as well as their impact on rhizosphere and soil microbial communities are needed to guarantee safe and reliable application, independent of whether they are genetically modified or not. The first and most crucial prerequisite for effective use of PGPRs is that strain identity and activity are continuously confirmed. A combination of both classical and molecular techniques must be perfected for more effective monitoring of inoculants strain (both genetically modified and unmodified) after release into the soil. Recent developments in techniques for studying rhizobacterial communities and detection and tracking systems of inoculated bacteria are important in future application and assessment of effectiveness and consistent performance of microbial inoculants in crop production and protection.
Farah Ahmad, Fohad Mabood Husain, Iqbal Ahmad

Chapter 15. Pesticide Interactions with Soil Microflora: Importance in Bioremediation

Modern application of insecticides belonging to different chemical families to boost agricultural productivity has led to their accumulation in soils to levels that affect, directly and indirectly, soil enzyme activities and physiol-ogical characteristics of nontarget soil microflora including plant growth-promoting rhizobacteria, and, consequently the performance of crop plants. Various biological strategies can be applied for removing toxic substances, including insecticides, from the environment and are collectively known as bioremediation. Among biological approaches, the use of microbes with degradative ability is considered the most efficient and cost-effective option to clean pesticide-contaminated sites. The present review focuses on the role of naturally occurring rhizosphere microbes involved in degradation or transformation of insecticides.
Munees Ahemad, Mohammad Saghir Khan

Chapter 16. Baculovirus Pesticides: Present State and Future Perspectives

Baculoviruses pesticides are ideal tools in integrated pest management programs as they are usually highly specific to their host insects; thus, they do not affect other arthropods including pest predators and parasitoids. They are also safe to vertebrates and plants and to the biosphere. Over 50 baculovirus products have been used against different insect pests worldwide, and all have been produced in vivo, mostly on insects reared on artificial diets. However, there are cases of significant viral production in the field by applying a baculovirus against natural populations of the insect host and collecting dead or moribund larvae for further processing into a formulated product. Despite the considerable number of programs worldwide utilizing baculoviruses as biopesticides, their use is still low compared to another biological insecticide based on the bacterium Bacillus thuringiensis Berliner. As of the present, there are no programs using in vitro commercial production of baculovirus due to several technical limitations, and further developments in this area are much needed. Use of the baculovirus of the velvetbean caterpillar in Brazil has experienced a setback over the past 7 years due to modifications in cultural practices by soybean growers. Slow speed of kill by viral pesticides is a limitation that has led to considerable research effort toward developing faster killing agents through genetic modifications by either deleting or inserting toxin genes from scorpions and spiders into their genomes. However, these GMOs have not been used in practice due to significant resistance by the public to modified baculovirus genomes. Effective public extension services and farmer education toward application of biopesticides are much needed to expand the use of these products worldwide.
Flavio Moscardi, Marlinda Lobo de Souza, Maria Elita Batista de Castro, Mauricio Lara Moscardi, Boguslaw Szewczyk

Chapter 17. Fungal Bioinoculants for Plant Disease Management

Plant diseases are among the major constraints in the production of food crops and inflict significant losses to global agriculture. Pesticides are widely used to control plant diseases but their application is costly and, in some cases, may bring more disadvantages than benefits. Use of bioinoculants to control plant diseases is an economically viable and ecologically sustainable method of disease management. A large number of bioinoculants is available; among them, bioinoculant fungi constitute the majority and are widely used in different cropping systems. Important bioinoculants that directly parasitize plant pathogens include Trichoderma spp., Paecelomyces lilacinus, and Pochonia chlamydosporia. Plant growth-promoting fungi such as Aspergillus spp. and Penicillium spp. may also suppress plant pathogens. In general, bioinoculants are effective against seed- and soil-borne fungi and nematodes. However, an important limitation in their commercial use in crop protection is nonavailability of efficient immobilizing systems for delivery and survival of bioinoculants. This chapter describes important bioinoculants, their effects, and their mechanisms of action against plant diseases caused by fungi, bacteria, and nematodes. State-of-the-art technology available for the production of commercial formulation of bioinoculants, along with important lacuna, is also discussed.
Mujeebur Rahman Khan, M. Arshad Anwer

Chapter 18. Mycorrhizal Inoculants: Progress in Inoculant Production Technology

Of the seven types of mycorrhizae, the symbiotic association of plants with arbuscular mycorrhizae (AM) and ectomycorrhiza (ECM) is the most abundant and widespread. Mycorrhizal inoculant technology, especially of AM and ECM, appears to be a promising avenue for sustainable agriculture and forestry because of their extensive and productive association with plants. Production of mycorrhizal inocula is a complex procedure that requires commercial enterprises to develop the necessary biotechnological skill and ability to respond to legal, ethical, educational, and commercial requirements. At present, commercial mycorrhizal inocula are produced in pots, nursery plots, containers with different substrates and plants, and aeroponic systems, and by nutrient film technique, or in vitro. Different formulated products are now marketed, which creates the need for the establishment of standards for widely accepted quality control. Generally, preparation and formulation of mycorrhizal inocula are carried out by applying polymer materials with well-established characteristics and which are useful for agriculture and forestry. The most commonly used methods involve entrapment of fungal materials in natural polysaccharide gels, which includes immobilization of mycorrhizal root pieces, vesicles, and spores, in some cases coentrapped with other plant-beneficial microorganisms. Efforts should be devoted toward registration procedures of mycorrhizal inoculants to stimulate the development of mycorrhizal products industry. Biotechnology research and development in such activities must be encouraged, particularly with regard to interactions of mycorrhizal fungi with other rhizosphere microbes, and selection of new plant varieties with enhanced mycorrhizal traits to provide maximum benefits to agriculture and forestry.
Zaki A. Siddiqui, Ryota Kataoka


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