eChapters

Introduction and History

Leguminosae/Fabaceae is a superfamily of dicotyledonous plants, which in species diversity is the third largest family of plants accounting for 5% of Earth’s plant biodiversity and make up for more than at least 1% of the total Earth plant biomass. Among these, legume grains and pulses having more than 20 to 30% lysine protein content are cultivated in 145 million ha of land in developing countries. This family is also characterized by presence of specialized tumor like structure formation (nodules) on roots (occasionally on stem as in Sesbania rostrata, Aeschynomene) that is inhabited by microsymbiont gram-negative bacteria (Rhizobium), engaged in fixing atmospheric nitrogen or symbiotic biological nitrogen fixation.

Modern agriculture faces the challenge of increasing food production while minimizing environmental impacts like soil degradation, water pollution, and biodiversity loss. To address these issues, integrating microbial biotechnology with nanotechnology has emerged as a promising approach. Nanoparticles (NPs) with plants and microbes, enhances nutrient uptake, plant growth, and stress tolerance. Bioinoculants, which contain beneficial microorganisms, promote plant health, but are limited by stability and environmental factors. The synergistic use of nanoparticles and bioinoculant offers a novel solution, improving crop yield, nutrient uptake, and resilience to stress, reducing dependence on chemical inputs. This integration supports the development of more resilient, resource-efficient, and environment-friendly agricultural systems.

Abstract

Extensive occurrence of alkaline soils has been reported from the Indo-Gangetic plains of Northern India, which is commonly known as ‘usar’ or ‘reh’. High alkalinity and high exchangeable sodium percentage (ESP) of such soil render it inhospitable for normal crop production. Research efforts over the past few decades have resulted in evolving a number of practices for the reclamation and management of such soils. However, application of biological inputs in combination with some moderately salt tolerant plant species seems to be a better option. AM fungi is well known to enhance the ability of plants to cope with environmental stresses generally prevalent in the degraded ecosystems and stress conditions by providing a number of nutritional and non-nutritional benefits. Since isolates of AM fungi differ in their tolerance to adverse physical and chemical conditions in soil and are better adapted to their native stressed microhabitat, selection of appropriate native AMF isolate is necessary for any large scale reclamation program. Taxonomic diversity of AM fungi in the alkaline soils of Upper Gangetic plains of district Allahabad and adjoining areas has been investigated and it was found that such soils have a detrimental effect on the AM spore population, distribution and diversity. Maximum spore population occurred in winter and minimum in summer. Glomus followed by Acaulospora was the most dominant genus. Acaulospora scrobiculata and Glomus fasciculatum were the most frequent species followed by Glomus mosseae and Glomus aggregatum. Glomus fasciculatum was the most abundant species followed by Glomus mosseae and Acaulospora longula.

Abstract

Mycorrhizal symbiosis is a highly evolved mutually beneficial relationship found between Arbuscular Mycorrhizal Fungi (AMF) and vascular plants. Knowledge of taxonomic and functional diversity of AM fungi is important in understanding the biology and ecology of such a widespread mycorrhizal symbiosis. Classification and identification of AM fungi remains in the formative stages of growth, with all the attendant turmoil. Since Glomus is known to be an important AM genera for providing multifacet benefit to several economically important plants. Hence, in this review article an attempt has been made to describe 120 Glomus species along with the line diagram of 63 Glomus species for their proper identification that can be used as an identification manual for mycorrhizal researchers.

Abstract

Based on oxygen requirement, microorganisms have been classified into different groups, like facultative anaerobes, micro-aerophilic, aerotolerant and strict/obligate anaerobes. Obligate or strict anaerobes are the key components of anoxic environments and provide valuable ecosystem services like methanogenesis, sulphate reduction, metal reduction, degradation of pollutants etc. Apart from being valuable for the ecosystem, they are causative agents of human infections. They are the key players of human-gut microbiology, and can be used in bio-toilets, solid waste management, anaerobic digestion of waste, wastewater treatment processes and generation of waste to energy etc. Only few anaerobic microorganisms are pathogenic in nature. A mechanistic understanding about physiology and metabolism of anaerobic microorganisms is imperative for their industrial applications. Cultivation of these organisms is the first and very important step for the basic and applied study of anaerobes. Unlike cultivation of aerobic microorganisms anaerobic cultivation needs special care and facilities for successful handling of anaerobes. In the current chapter, We tried to incorporate the components and technicalities of anaerobic pre-reduced media preparation, cultivation and successful isolation of strict anaerobes. In addition, we also tried to discuss the different tools used for cultivation of anaerobes.

1. Introduction 
The Techno-Economic Analysis (TEA) of nanotechnology-based microalgal biodiesel explores the intersection of nanotechnology and renewable energy production, aiming to assess the economic feasibility and sustainability of integrating nanomaterials into the microalgal biodiesel production process (BlancoCanqui, 2010). As the global demand for sustainable energy sources continues to rise, microalgae have emerged as a promising feedstock for biodiesel production due to their high lipid content and rapid growth rates. Nanotechnology offers innovative solutions to enhance various stages of the microalgal biodiesel production chain, from cultivation and harvesting to lipid extraction and conversion (Moustakas et al., 2020).This introduction provides an overview of the key objectives, methodologies, and implications of conducting a TEA on nanotechnology-based microalgal biodiesel. It outlines the significance of integrating nanomaterials into biodiesel production processes, highlights the potential benefits and challenges associated with nanotechnology adoption, and sets the stage for the subsequent analysis of economic factors and technical considerations (Borowitzka and Moheimani, 2013).The introduction also discusses the broader context of sustainable energy transition and the role of microalgal biodiesel as a renewable alternative to conventional fossil fuels (Gaurav et al., 2017). It emphasizes the importance of evaluating not only the technical feasibility but also the economic viability and environmental sustainability of nanotechnology-enabled biodiesel production methods. By conducting a comprehensive TEA, this study aims to provide valuable insights into the cost-effectiveness, scalability, and market potential of nanotechnology-based microalgal biodiesel, informing strategic decision-making and investment priorities in the renewable energy sector (Gouveia and Oliveira, 2009; McCollum et al., 2018). Through interdisciplinary collaboration and rigorous analysis, the TEA seeks to advance our understanding of the economic and technological factors shaping the future of sustainable biofuel production.

4.1 Introduction 
The growing recognition of biofuels as a competitive renewable energy source is increasingly evident, particularly when compared to the fossil fuel energy's limited supply, unstable geopolitical environment, and environmental repercussions associated with fossil fuel energy. In the broader context of renewable energy sources (RES), which are characterized as sustainable resources long-time availability at an affordable price and devoid of adverse effects, biofuels emerge as a promising category.(Charters, 2001) Biofuels include any type of energy-dense material that is either directly acquired from biological activities or is obtained through the chemical conversion of biomass from previous species.(Rodionova et al., 2017) Because biomass can be effectively converted into energy, biofuels play a crucial role in supplying energy needs and consumptions. The importance of biofuels in the global energy landscape is underscored by several factors, making them relevant technologies for both developing and industrialized countries.(Demirbas, 2007) Fuel, a concept central to energy discourse, encompasses substances that store and release energy when subjected to a chemical reaction. It serves as the lifeblood of energy systems, powering various sectors from transportation to electricity generation. The variety of fuels available is staggering; they include traditional petroleum-based fuels such as coal, oil, and natural gas, in addition to renewable energy resources like solar and wind and, most crucially, biofuels and alternative energy sources like nuclear power.(M et al., 2023) Within the realm of biofuels, there exists a multifaceted spectrum, where each type offers unique advantages and applications. Bioethanol, derived primarily from crops such as corn and sugarcane, stands out as a prominent example. Biodiesel, synthesized from vegetable oils or animal fats, represents another significant category. Additionally, advanced biofuels, produced through cutting-edge technologies like cellulosic ethanol and algae-based biofuels, showcase the ongoing innovation within the biofuel industry.(Vickram et al., 2023) There are a number of reasons why people choose biofuels over conventional fuels, but their favorable effects on the environment are the main one. Unlike fossil fuels, biofuels present a renewable and sustainable option, as they are derived from organic materials that could be replenished. This contrasts starkly with the finite nature of fossil fuel resources, which are not only depleting but also contribute to ecological degradation. Moreover, the instability in the geopolitical landscape surrounding fossil fuels, with its attendant economic and security implications, further accentuates the appeal of biofuels as a more secure and resilient energy source.(Lee, Clark and Devereaux, 2008) When

Introduction

Antibiotics are the substances produced by one microorganism and are required to inhibit the growth of other microorganisms. Antibiotics are used to combat infectious diseases. The antibiotic sensitivity is very significant due to development of resistance among various microorganisms.

Requirements

1. Swab
2. Nutrient agar
3. Petri plates
4. Bacterial cultures
5. Forceps
6. Antibiotic discs
7. BOD Incubator

Procedure

1. Prepare the bacterial lawn on Nutrient agar plate.

Introduction
Some species of rod shaped bacteria e.g. E.coli, Ps. fluorescence, Bacillus subtilis are known to be motile. Bacterial motility is generally associated with the presence of hair-like appendages called “Flagella”. There are several ways for determining the motility of a culture such as the wet mount method, hanging drop method and a culture method (on semisolid medium). Bacterial cells in suspension also exhibit a vibratory or oscillatory movement without changing their relative position. This is termed ’Brownian Movement’ and it should not be confused for true motility of bacteria. In motility the cells change their relative position and move across the microscopic field in different directions.
1. The Wet Mount Technique
This is the simplest and quickest way to determine the motility of bacteria.
Materials
1. Clean glass slide
2. Broth culture of bacteria (E.coli, Ps.fluroscenes)
3. Cover glass
Procedure
1. Place a drop of organisms on clean glass slide and cover with a cover glass.
2. Examine the slide with phase contrast microscope or bright field microscope with reduced light under oil-immersion.

With increasing industrialization, water sources available for consumption as potable water are being adulterated with industrial, animal and human wastes. As a result water has become a formidable factor in disease transmission. Polluted water usually contains large amount of organic matter which forms nutritional base for growth and multiplication of microorganisms. Usually the water with non-pathogenic organisms, like autotrophs and saprophytic heterotrophs, are not of major concern, but presence of intestinal contaminants of fecal origin are of great concern. These pathogens are responsible for intestinal infection such as bacillary dysentery, typhoid fever, cholera and paratyphoid fever.

The detection of all the intestinal pathogens on a routine basis would be a tedious and difficult task, however, it would be easy to demonstrate the presence of some of the nonpathogenic intestinal organisms like Escherichia coli and Streptococcus faecalis. These are always found in intestine but their detection confirms the contamination of water with fecal material. Such organisms are also called pollution indicator.

Abstract

The normal human microbial flora comprises of both aerobes and anaerobes at various sites. Anaerobes are recognised as pathogens causing simple abscesses to life threatening human infections like gas gangrene. They are also incriminated in a large number of mixed bacterial infections and their diagnosis and specific therapy is important for better patient outcomes. The emergence of Clostridioides difficile as an important pathogen causing antibiotic associated diarrhoeas and its emerging resistance to antimicrobial agents has brought anaerobic microbiology into the forefront. Unfortunately, anaerobic clinical microbiology is difficult to practice with long turnaround time, difficult to grow pathogens, new genera and species being discovered based on 16S rRNA sequencing methods and increasing incidence of antimicrobial resistance being reported in anaerobes. This chapter delves into the important aspects required to improve utilization of anaerobic microbiology services and the variety of human infections we encounter in clinical practice due to anaerobic bacteria.

Fungi are very primitive eukaryotic microbe on the earth which are evolved with the earth and its atmosphere. These may be the ancestor of the plants in the process of evolution (Borkar, 2001). The group of ancestral fungi is thought to be represented by the present day chytridiomycota, although the Microsporidia may be an equally ancient sister group. The first major steps in the evolution of higher fungi were the loss of the chytrid flagellum and the development of branching, aseptate fungal filament, which occurred as terrestrial fungi diverged from water molds 600 million to 800 million years ago. The easily recognizable mushroom fungi probably diversified 130 million to 200 million years ago, soon after flowering plants became an important part of the flora and well before the age of dinosaurs. A relatively recent evolutionary radiation perhaps 60 million to 80 million years ago, of anaerobic chytridiomycota occurred as grasses and grazing mammals became abundant, the chytrid fungi serve as symbionts within the rumen of such animals, thereby enabling the grazing mammals to digest grasses.

The fungi are present everywhere. They are in the soil and soil crust, in atmospheric air, in sea and pond water, rivers and water streams, and on the surfaces of almost all the material ,whether living or otherwise, on affected food stuff , vegetables and food grains, on body of human beings, animals, birds and fishes, on plant surfaces, on leathers and clothes in damp weather, on house walls and ceiling in damp conditions, on railway coaches and buses roofs having water leakage and dampness, in caves, on garbage and decomposing material etc. The fungal spores are present everywhere, however the fungal growth occurs where the congenial conditions for their growth is available.

ABSTRACT

Industrial and agricultural activities and the disposal of waste products have resulted in the contamination of many terrestrial environments. Industrial effluents often contain considerable amount of potentially toxic compounds such as Cd, As, Pb and Hg. Due to their small size, which provides a large contact area that can interact with the surrounding environment, microorganisms are the first biota showing the impact of toxic compounds. Microorganisms being in intimate contact with the soil environment are considered to be the best indicators of soil pollution. In general, they are very sensitive to low concentrations of contaminants and provide a rapid response to soil perturbation. Rhizobium spp. are ubiquitous gram-negative soil bacteria that have a profound scientific and agronomic significance due to their ability to establish nitrogen-fixing symbiosis with legumes, which is of major importance to the maintenance of soil fertility and to feed livestock and populations in many developing countries. Therefore, the present work aimed to evaluate the impact of heavy metals on Rhizobium populations isolated from different locations contaminated with heavy metals. In order to reach this goal some physicochemical parameters that influence metal bioavailability were determined, as well as their concentrations in soil. Cadmium tolerance of Rhizobium isolates was screened in artificial media and the highest cadmium concentration allowing growth was defined as the maximum resistance level (MRL). The heavy metal tolerance variability found among Rhizobium isolates, suggests that beyond basic metabolic machinery also exist variations that allow isolates to display distinct tolerance responses to Cd stress. To better understand Rhizobium cadmium tolerance, wall-bound and intracellular Cd was determined. Sensitive and moderately tolerant isolates showed low intracellular Cd amounts when compared to extremely tolerant isolates, which were able to withstand intracellular Cd concentrations 40 times higher. Lipopolysaccharides (LPS) bind cations unspecifically, so they constitute the initial defence mechanism to heavy metals since they sequestrate cations and therefore prevent their entry into the cell. LPS quantity varied significantly between isolates. LPS increases were observed in most sensitive, moderately tolerant and tolerant isolates, evidencing an attempt of cells to adjust to cadmium entry. These results may be of great importance for understanding how Rhizobium persists in contaminated soils. They also provide important data to the development of bioremediation processes and to the improvement of soil productivity.

6.1 Introduction 
Lignocellulosic Biomass (LCB) generated from the global consumption of fruit and vegetables rapidly generates approximately 140 billion metric tons of agricultural waste. Environmental pollution associated with these wastes, like land pollution, and increased greenhouse gas emissions, can be reduced through the bioconversion of wastes (renewable biomass resources) into value-added products in biorefineries (Babu et al., 2022). These value-added products synthesized for utilization in industries such as biofuels, biomaterials, or biochemicals are effective, reuseable, and enhance sustainability. These bioproducts are recuperated into useful biomolecules and oils for the food industries and dyes for the textile industries. The sustainability of these products can be acquired efficiently through costeffective pretreatments and bioprocess optimization methods (Haldar et al., 2020). The initial stage of value product chain development is the process modelling and optimizing, which, due to its complex nature and large scale, faces challenges in raw material availability, cost competitiveness, upscaling, and lack of technological maturity (Gerassimidou et al., 2021). Optimization of production processes like fermentation and chemical synthesis for manufacturing value-added products has become a focal point in research enhancement of biorefineries. There has been a revolution in the technology used in process designing and manufacturing practices in biorefineries. Advanced Artificial Intelligence (AI)-based approaches like Genetic Algorithm (GA), Fuzzy Inference System (FIS), and Artificial Neural Network (ANN) are being utilized along with conventional tools to optimize variables interacting in production processes. These techniques increase overall efficiency and enhance prediction accuracy and output of value-added products (Banerjee et al., 2025). Optimization of the bio processes enables higher productivity in parallel with lower environmental burdens. AI-based approaches integrated with conventional approaches need a large amount of experimental historical data to be successfully carried out (Arias et al., 2023). Advancement of biorefineries due to the rapid demand of value-added products as well as a solution to the greenhouse emissions, the integrated application of AI for process analysis needs to be understood. This chapter is a contribution towards upscaling the current knowledge base related to different approaches for the enhancement of production for value-added products. The chapter discusses the conventional techniques for value-added production and various optimization models. There is further discussion of advancements in the traditional approach using AI-based approaches and their features and benefits in biorefinery. Apart from different AI-based models, AI integrated with life cycle analysis for sustainability is also briefed. Finally, the chapter addresses current limitations in the field and outlines potential future directions for research and development.

5.1 Introduction
Bioprocess engineering is becoming more advanced form of technology due to the many changes taking place in the field. The world is in dire need for creative, well thought out, and innovative approaches to problems associated with sustainable biomanufacturing, treatment customization, and the production of bio-based goods. One of the most notable advancements is the rise of synthetic biology, which allows for bioproduction at unprecedented levels where microbes could be modified for better bioproduction. More traditional approaches is strain improvement and metabolic engineering, which revolve around mutation-based processes and other forms of gene transfer technologies. The applications of new, non-traditional methods such as machine learning and artificial intelligence have completely revolutionized the way bioprocessing and its monitoring, as well as optimization, is done. On top of that, the drive towards sustainability has further motivated the use of green bioprocessing techniques which aim to minimize environmental harm. In parallel with these advances, regenerative medicine and gene treatment is opening doors to a new era of personalized medicine. In a world where change is constant, these accomplishments and innovations help us to take full advantage of what bioprocess engineering has to offer, as well as face the intricate challenges that the modern world encounters (Awasthi, 2022). In recent years, the field of biotechnology has undergone transformative and profound change that is marked by new breakthroughs and improvement which can affect several fields such as medicine, agriculture, and ecology. The emergence of tools like CRISPR-Cas9, synthetic biology, bioprinting, precision medicine and regenerative medicine are some of the new trends in biotechnology. These changes are recognizing the limits on the biological sciences and expanding them at the same time. Synthetic biology is another technology that includes the strategy and production of new genomic parts, related tools, and re-designs of traditional ones for valuable purposes. The scientists are modifying living organisms to perform useful functions like manufacturing biofuels or pharmaceuticals. Synthetic biology raises concerns about biosecurity and the unintentional release of artificial organisms into the environment, despite its enormous potential to address global challenges like renewable energy and sustainable resource usage (Jain, 2022). This article introduces advancements in bioprocess engineering with specific focus on advancements brought about by emergence of CRISPR. Bioprocess engineering is the application of process engineering principles to biological systems and has been a multidisciplinary field unto itself. Since its isolation as a groundbreaking genome-editing system, CRISPR has vastly improved the precision, efficiency, and scalability of both microbial and mammalian cell manipulation. This has resulted in numerous breakthroughs in pharmaceuticals, biofuels, and industrial enzymes, a major milestone for synthetic biology, and metabolic engineering (Jinek et al., 2012). The CRISPR revolution has changed

Abstract

Drought and salinity are two major abiotic stresses that affect various aspects of human lives of one third world population including human health and agricultural productivity. Abiotic stresses such as salinity, drought, nutrient deficiency or toxicity, and flooding limit crop productivity world-wide. A living organism is considered resistant if it tolerates a given physicochemical stress. The presence of microorganisms within the soil, specially a main group known as arbuscular mycorrhizal fungi (AMF), is a key factor in the adaptation of plants to the different ecosystems. The AMF colonizing within the plant roots can determine the success of plants in salt- and/or drought-affected areas. The symbiosis with AMF has been proposed as one of the mechanisms of salinity and water stress avoidance. Studies have shown greater drought and salinity tolerance in AMF-colonized plants by a number of mechanisms. These mechanisms include enhanced water and nutrient uptake directly by extraradical hyphae, higher leaf stomatal conductance and/or better root system architecture, higher capacity of osmotic adjustment and antioxidant defense systems, better soil structure due to higher glomalin and over expression of genes encoding antioxidant enzymes.

Introduction
The eco-friendly and commercially sustainable production has been a crucial aspect of modern commercial development. The ecologically sustainable microbial production of value-added products has seen rapid growth across multiple sectors, such as food, pharmaceuticals, and energy, resulting in notable economic benefits and social advancements on a global scale (Shi et al., 2022). The development of “omics” technologies has aided the improvement of microbial production processes through diverse omics procedures, such as genomics, transcriptomics, metabolomics, and systems biology (Amer and Baidoo, 2021). These omics approaches give important understandings into the widespread metabolic variations, thereby identifying the factors that affect microbial production efficiency and improving the understanding of targeted optimization strategies. The integration of omics approaches is considered essential for any strategies that aim to be industrially sustainable. Industrial activities depend on the cultivation and accessibility of significantly competent microbial species to optimize bioproduct yields (Keasling, 2012). Recent progress in the industrial generation of value-added products heavily depends on strategies rooted in omics. Typically, the industry can achieve cost-effective production of limited quantities of biopharmaceuticals (Harris et al., 2015). Nevertheless, to sustainably scale up production capacities, economically feasible alternatives must be planned, standardised, and developed. Over the past few decades, microbes with enhanced abilities for superior product generation have been identified (Babar et al., 2018; Kim et al., 2015). Latest improvements in metabolic engineering have aided the manipulation of various genetic components, including coding and regulatory regions, to maximize commercial benefits (Babar et al., 2018). Genomic methodologies aim to investigate the variations among individuals at both the germline and somatic levels by sequencing specific genomes. The transition from DNA microarray technology to DNA sequencing has enabled the thorough sequencing of entire genomes, providing a detailed characterization of an organism’s genomic landscape (Dai and Shen, 2022) whereas, transcriptomics involves the analysis of mRNA, microRNAs, long non-coding RNAs, and circular RNAs. The methods used in transcriptomics are designed to identify and enumerate RNA molecules transcribed from a specific genome at a particular time. Proteomics on the other hand focuses on understanding the functional significance of all proteins expressed within a cell, tissue, or organism by examining the flow of information via protein signalling and metabolomics contributed a vital part in the development of bioprocesses by providing a comprehensive overview of metabolic activities (Horgan and Kenny, 2011; Petricoin et al., 2002). Synthetic biology contributes a vital part in the production of high-performance strains for microbial manufacturing. A variety of microorganisms including bacteria, fungi, and microalgae can be employed in microbial production. However, synthetic biology methods primarily emphasis on the balanced blueprint of biosynthetic metabolisms for target outcome, as well as the creation and standardisation of these pathways and the heterologous production of the biosynthetic components (Wan et al., 2023). These approaches include the optimisation of fermentation process, optimisation of medium composition, scale up of fermentation, optimization of cell growth for manufacture of desired product, and strategies to decrease feedback inhibition and product toxicity (Figure 3.1) (Wan et al., 2023; Zhang et al., 2022).

7.1 Introduction 
The global economy of today magnifies the extend and scope of product safety issues for a wide range of items, from food and medicine to consumer goods, medical devices, and automobiles (Marucheck et al., 2011). Industrial globalization has raised awareness of the many risks and vulnerabilities that goods face as they move through the supply chain, from design and sourcing to manufacturing, shipping, distribution, and ultimate sale to the end user (Milovanovi? et al., 2017). These challenges necessitate the implementation of reliable and sustainable methods for producing goods. This can be achieved via biorefineries, which may reduce supply chain disruptions while promoting environmentally friendly resource utilization. Biorefineries are usually referred to as first and second generation biorefineries with accordance to the raw material utilized. The former uses food crop as raw material wherein the latter utilizes lignocellulosic and agricultural residues as raw material for generation of value-added products (Guo et al., 2015). Additionally, algae biomass is utilized in the third generation biorefineries (Parajuli et al., 2015). However, among these three the first generation are the most established while the second and third are still developing at various stages due to technical and economic challenges (Gerssen-Gondelach et al., 2014). Globally, ethanol biorefinery processing U.S. corn and Brazilian sugarcane dominate production contributing 52% and 28%, respectively (Renewable Fuels Association, 2023). These biorefineries are further distinguished based on categories such as sugars, oils and syngas which serve as key intermediates in production of bioethanol, glycerol or lactic acid (Calvo-Flores and Martin- Martinez, 2022). Additionally, these biorefineries utilizes feedstock and residues which can be derived from forest, sugar crops and oil crops. The processing technologies employing for utilization of such raw material are fermentation, gasification and pyrolysis. This configuration of biorefinery determines the integration and systematic pathway from raw feedstock to final product (Aristizábal?Marulanda et al., 2019). Furthermore, both the supply chain and value chain are significant part of biorefineries. The value chain stands for the value-oriented aspect of the process which is often derived by customer demand and include product innovation, marketing and value development (Sharma et al., 2013). In contrast, the supply chain focuses on the logistical flow of materials, encompassing the movement of raw materials from suppliers to end consumers. However, certain stages of supply chain overlap with biorefinery systems such as separate pretreatment and fermentation facilities (Sutduean et al., 2019). The design of biorefinery system involves a structural decision process at strategic, tactical and operational levels. These decisions are made with respect to various biohazards at each level. The macro-level strategic decisions, address long-term risk mitigating strategies to ensure overall safety (Chaturvedi et al., 2020). Once the high-level decisions are established, tactical and operational decisions are implemented. The tactical decisions focus on implementing safety measures with processes such as handling hazardous materials and optimizing biorefinery. While

Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) technique, invented in 1985 by Kary B. Mullis, allowed scientists to make millions of copies of a scarce sample of DNA. The technique has revolutionized many aspects of current research, including the diagnosis of genetic defects and the detection of the AIDS virus in human cells. The technique is also used by criminologists to link specific persons to samples of blood or hair via DNA comparison. PCR also affected evolutionary studies because large quantities of DNA can be manufactured from fossils containing but trace amounts.

Kary Mullis invented the PCR technique in 1985 while working as a chemist at the Cetus Corporation, a biotechnology firm in Emeryville, California. The procedure requires placing a small amount of the DNA containing the desired gene into a test tube. A large batch of loose nucleotides, which link into exact copies of the original gene, is also added to the tube. A pair of synthesized short DNA segments, that match segments on each side of the desired gene, is added. These “primers” find the right portion of the DNA, and serve as starting points for DNA copying. When the enzyme Thermus aquaticus (Taq) is added, the loose nucleotides lock into a DNA sequence dictated by the sequence of that target gene located between the two primers.

The test tube is heated, and the DNA’s double helix separates into two strands. The DNA sequence of each strand of the helix is thus exposed and as the temperature is lowered the primers automatically bind to their complementary portions of the DNA sample. At the same time, the enzyme links the loose nucleotides to the primer and to each of the separated DNA strands in the appropriate sequence. The complete reaction, which takes approximately five minutes, results in two double helices containing the desired portion of the original. The heating and cooling is repeated, doubling the number of DNA copies. After thirty to forty cycles are completed a single copy of a piece of DNA can be multiplied to hundreds of millions.