eChapters

ABSTRACT
Rice straw is one of the most abundant lignocellulosic agricultural wastes in India and can be used as a major resource for biogas production. The structure of rice straw is complex and is made up mainly of cellulose, hemicelluloses and lignin. The lignin physically shields the cellulose and hemicelluloses, the main components which can be converted to energy, and hinders the access of microbial enzymes to these carbohydrate moieties. To overcome this making the straw a potential resource of biogas production, effective pretreatment is necessary that can efficiently hydrolyse the straw and make it accessible to cellulolytic enzymes and methanogens. Various pretreatment methods which are employed currently in the market are discussed here. For a process to be economically feasible it is necessary that the pretreatment be efficient as well as cost effective. To gain some insights into this aspect, the techno-commercial feasibility of Biomethanation of rice straw has been discussed.

ABSTRACT
Recent challenges for fossil fuels, associated environmental issues with disposal of organic garbage, and the rising costs for energy has encouraged researchers to search for alternate energy sources. Massive uncontrolled biogas from landfills, sludge and organic material degradation under anoxic conditions is an environmental management issue. Methane emission to atmosphere has a tremendous green house effect, but this can also be a valuable renewable energy resource. Biogas from kitchen wastes through anaerobic digestion by methanogens is such an alternate technology. Kitchen wastes are considered one of the best raw materials for biogasification. Biogas is mainly composed of 50-74% methane (CH₄), 25-28% carbon-di-oxide (CO₂), other volatile compounds including hydrogen sulphide (H₂S), water vapour and other trace gas compounds. The impurities in it, like Hydrogen sulphide (H₂S) which is corrosive, downgrade its quality. Purified biogas of higher calorific value attracts attention as a substitute fuel for additional applications. Its purification using various suitable scalable and economically-viable scrubbing technologies to enhance its quality and performance as an alternate fuel is an important step in this direction. Thus, a technology to generate and purify biogas from kitchen waste has significant prospects in the energy sector, and also as a promising enterprise to employ the youth.

ABSTRACT
Increased environmental pollution and high demand over supply for conventional fossil fuel sources have led to the exploration of other renewable energy sources. Out of the potential alternatives, methane has come up with a promising unconventional fuel source. Several technologies have been developed to establish methanation, including biomethanation, chemical methanation and gas-to-liquid technology. Several factors like process parameters, catalysts and reactors have been studied for process optimisation and making methane production commercially viable. Although methane as a greenhouse gas is a contributor to global warming, its combustion produces relatively ‘environment friendly’ gas CO₂, and H₂O. The released CO₂ is fixed into biomass by plant through photosynthesis process. Only rarely does methane combustion produces CO when combusted in an oxygen-limited condition. Thus, methane production and combustion are carbon neutral process. This chapter focuses on the application of methane as a source of alternate energy. Various technologies used, recent advances and challenges faced in methanation process are discussed.

ABSTRACT
Biomethanation comprises of four interdependent stages in series, hydrolysis, acidogenesis, acetogenesis and methanogenesis. Methanogenesis, the final stage of biomethanation, is accompanied with the reduction of substrates that include organic acids such as CH₃COOH, and others such as CO₂ and CH₃OH to CH₄ by methanogenic and methanotrophic archaea, usually under anaerobic conditions. Strictly anaerobic methanogenic archaea are found in the ruminant intestine, sewage digester, groundwater and deep soil/water etc. In methanogenesis, H₂ acts as the electron donor while substrates CO₂, CH₃OH and probably CoM-S-S-CoB (for CH₃COOH) act as terminal electron acceptors and reduce to methane, a process known as anaerobic respiration. This conversion is associated with the electrogenic proton motive force along with sodium motive force to drive the ATP synthesis by A0A1 ATP synthase, known as respiratory phosphorylation. The mechanism of conversion of methane from CO₂ by methanogens with cytochromes is same as those without cytochromes except that the reduction of heterodisulphide CoM-S-S-CoB is performed by the membrane-bound VhoACG-HdrDE complex for former while by cytoplasmic MvhADG–HdrABC complex for the later.

ABSTRACT
Methanogenesis is an important biogeochemical process takes place in anaerobic ecological sites. It is the last step of terminal electron accepting process of anaerobic respiration. Methanogens synthesise CH₄ using acetate and CO₂ as precursor molecules. Former types of methanogens are referred as acetoclastic methanogens and the later ones as hydrogenotrophic methanogens. After biogenesis of CH₄, it is emitted to atmosphere and causes global warming. In aerobic ecosystem CH₄ is oxidised by methanotrophs to CO₂ a less global warming molecule. However, in anoxic environment CH4 acts as an electron donor and interacts with various electron acceptors. Methane modulates denitrification, Fe reduction, SO₄ reduction and organic matter decomposition. Manuscript elucidates methanogenesis and highlights the pathways of interaction between methanogenesis and cycling of electron acceptors in various anaerobic ecosystems in relation to global C and N balance.

ABSTRACT
Methanogens are primarily anaerobic Archaebacteria representing the most primitive earthly creatures, alive since about 3.5 billion years. While oxygen inhibit their activity, they adapt well in a variety of ecological niche, viz., the intestinal tracts of insects and animals including ruminants, sewage digesters, groundwater and deep soil/water, etc. They are involved in biomethanation that converts organic polymers, including the complex recalcitrant lignocelluloses, to CH₄ and CO₂. Enhancing the process by optimising the physical, chemical and molecular parameters is an interesting biotechnology. Molecular research is rapidly progressing on the genomics, gene and its regulation, and methanogenesis as it expresses. Identifying novel species expressing methanogenesis and manipulating the genetic make-up including through cloning in recent years will set goals and leads for future investigations. The biomethanation enzyme Methyl-CoM-reductase (Mcr) constitutes about 10% of the methanogen protein. Due to its abundance and significance, elucidating its structure and its synthesis and regulation mechanism has received much attention recently. Mcr-coding genes from methanogens have been cloned and sequenced. An approach at the molecular level to enhance biomethanation seems to lie in metagenomics. The microbiology, biochemistry, the roles of the participating microbes, their molecular biology and applications in methanogenesis are discussed in this chapter.

ABSTRACT
Methanogenesis is a ubiquitous process primarily encountered in anaerobic environments. Methanogens are a group of diverse obligate anaerobic bacteria that produce methane from complex organic matter. Despite their differences in morphological characters and habitat, they show coherence in terms of methane-producing metabolic activity. Being distinctive from all other classical prokaryotes, these are grouped under another kingdom of Archea. Other distinctions of the members are based on their unique cell wall structures and membrane components, as well as differences in their highly conserved gene sequences. Methanogens likely owe their cosmopolitan status to their unique mode of energy metabolism of methane generation. They are active in various ecological niches, from rumens of animals to submarine volcanic vents, sewage digestors and landfills. Methanogenic bacteria are abundant in habitats where high affinity electron acceptors are negligible. Methane fermentation involves bacteria that obtain energy by catalysing anaerobically degradable organic matter to CH₄ and CO₂ by consuming H₂, CO₂, and low-carbon organic acids as their substrates. The gaseous products of methanogenesis are greenhouse gases, thus are major contributors to global warming. Methanogens could be used to produce renewable carbon-neutral energy substitute, biogas. Methane cycling archaea play a major role in global methane production as energy for the future.

1.Which one is the example of abiotic stress?

a.Drought

b.Salinity

c.Flood

d.All of these

2.Which one is the example of osmoticum?

a.NaCl

b.PEG

c.H₂O

Objective: To quantify aflatoxin content in given sample by Indirect competitive ELIS

Background information:

Enzyme linked immuno sorbent assay (ELISA) is a common biochemical technique that uses the principle of solid-phase enzyme immunoassay (EIA) for detection of substances mainly antigens in a liquid or wet sample.

ELISA is a very popular technique that is used for diagnostic purpose in health sector, plant protection and also for quality test in food and other industries. This technique applies the principle of antigen-antibody reaction on a solid surface. The attached antibody is linked to certain enzymes and in the end of reaction by addition of substrate that performs enzyme-substrate reaction and makes product that has some signal mostly color change and based on intensity of color the qualitative as well as quantitative estimation of antigens can be done using ELISA reader coupled with spectrophotometer.

In ELISA technique one antibody should be specific to certain antigen. For reaction of ELISA the ELISA plates are used that is also known as microtiter plate made-up of polystyrene. The sample to be tested for ELISA contain unknown amount of antigen to be detected, this sample containing antigen is immobiliz d on a solid support like polystyrene microtiter plate in two different ways; first is non-specific where antigens adsorbed to the surface and second is specific where the antigens of sample are captured by specific antibody to the antigen, like used in sandwich ELIS Once the antigens are immobilized non-specifically or specifically the detection antibody should be added that makes precise antigen-antibody complex. The detection antibody may be either covalently linked to certain enzyme or may be detected by another secondary antibody linked to bioconjugated enzyme. During ELISA steps several proteins or antibodies may non-specifically bound that can be removed by washing with mild detergent solution in between of each ELISA step. For washing of ELISA plates, ELISA plate washer may be used that mechanically wash the ELISA plates. At the end of ELISA the final wash is given and substrate is added that produces visible signal in the form of color change that indicates the presence of antigens in the sample and further quantification may be done using ELISA reader coupled with spectrophotometer.

Objective: To synthesize cDNA from isolated plant total RNA.

Background information:

Complementary DNA (cDNA) is double-stranded DNA made from template of single stranded RNA, [e.g., mRNA or microRNA] by reverse transcriptase enzyme. Mostly cDNA is used for cloning of eukaryotic genes in the prokaryotes. For the expression of a specific protein in a cell where the expression of that protein is not found (i.e., heterologous expression), the cDNA will be transferred that codes for the protein to the recipient cells. Retroviruses (e.g. HIV-1, HIV-2, simian immunodeficiency virus, etc.) also produce cDNA naturally and integrate in host's genome and further creates provirus (Croy, 1998). Major use of cDNA is in gene cloning or as gene probes or in the creation of a cDNA library. When scientists transfer a gene from one cell into another cell for the expression of new genetic material as a protein in the recipient cell, the cDNA will be added to the recipient (rather than the entire gene), because the DNA for an entire gene may have certain DNA that may not code for the protein or that interrupts the coding sequences of the protein (e.g., introns). Partial sequences of cDNAs are often obtained as expressed sequence tags.

Objective: To check the integrity of isolated RNA on agarose gel.

Background information:

The most common method used to assess the integrity of total RNA is to run RNA sample on agarose gel (denaturing) stained with ethidium bromide (EtBr). While native (non-denaturing) gels can be used, the results can be difficult to interpret. The secondary structure of RNA alters its migration pattern in nativegels so that it will not migrate according to its true size. Nondenaturing conditions also result in bands that are not as sharp, and even multiple bands representing different structures of a single RNA species.

Intact total RNA run on a denaturing gel will have sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit the 2:1 ratio of high quality RNA. Completely degraded RNA will appear as a smear (having very low mw). Inclusion of RNA size markers on the gel will determine the band size or smears size and will also serve as a good control for ensuring proper gel run. Note: Poly selected samples will not contain strong rRNA bands and will appear as a smear from approximately 6 kb to 0.5 kb (resulting from the population of mRNAs, and depending on exposure times and conditions), with the area between 1.5 and 2 kb being the most intense (this smear is sometimes apparent in total RNA samples as well).

Objective: To isolate RNA from plants using TRIzol reagent and its quantification by spectrophotometer.

Background information

RNA (Ribonucleic acid) is a polymeric substance present in living cells and many viruses, consisting of a long single-stranded chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine, and uracil, which are bonded to the ribose sugar. RNA is necessary in all the steps of protein synthesis in all living cells and carries the genetic information for many viruses.

The isolation of RNA with high quality is a crucial step required to perform various molecular biology experiment. TRIzol Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues (Chomczynski, 1993).TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components. Addition of chloroform, after the centrifugation, separates the solution into aqueous and organic phases. Aqueous phase contains RNA.

After transferring the aqueous phase, RNA can be recovered by precipitation with isopropyl alcohol. DNA and proteins can be recovered by sequential separation after the removal of aqueous phase. Ethanol precipitation needs DNA from the interphase, and an additional precipitation with isopropyl alcohol requires proteins from the organic phase. Total RNA extracted by TRIzol Reagent is free from the contamination of protein and DNA The isolated RNA can be further used in Northern blot analysis, in vitro translation, poly (A) selection, RNase protection assay and molecular cloning.

Objective:To transfer DNA on nylon membrane by Southern blotting.

Background information

A Southern blot is a method used in molecular biology to detect a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.

The British biologist Edwin Southern (Southern, 1975) discovered this technique the technique is known by his name. Southern blotting technique uses digested genomic DNA with restriction enzymes. Southern blotting technique may be used to identify the gene copies number in a genome. A probe that hybridizes only to a single DNA segment that is uncut with restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several highly similar sequences (e.g., may be due to sequence duplication).

In the southern blotting technique a short DNA probe is used that matches based on complementary DNA sequence fragment on filter membrane. It proves that the DNA fragment on membrane contain specific sequence for which DNA probe is prepared. When DNA is transferred on the membrane from gel it permits binding of radioactive/Biotin labeled hybridization probe to the restricted DNA. Southern blotting is also important for autoradiography.

Southern blotting is an important molecular biology technique that may be used for homology based cloning, screening a DNA library, screening of cloned DNA fragments and to identify methylated sites in genes.

Objective: To amplify DNA using RAPD primers and study on agarose gel.

Background information:

RAPD markers are decamer (10 nucleotide length) DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence and which are able to differentiate between genetically distinct individuals. RAPD is applied to analyze the genetic diversity by random primers. Unlike traditional PCR analysis, RAPD does not require any prior DNA sequence information of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions complementary with primers' sequence. For example, no fragment is produced if primers annealed too far apart or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in the templ that was earlier complementary with primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel. RAPD is a dominant marker means it cannot distinguish between heterozygous; single copy and homozygous; two copies. Rarely RAPD markers may be Co-dominant where different fragment length of DNA is amplified from the single locus. PCR is a molecular technique used for amplification of DNA, a complete enzyme based chemical reaction, therefore, the concentration and quality of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully developed laboratory protocols to be reproducible. Mismatches between the primer and the template may result in the total absence of PCR product or decreased amplified product. Thus, the RAPD results can be difficult to interpret.

Objective: To check quality of genomic DNA on agarose gel electrophoresis.

Background information

Agarose gel electrophoresis is a common method of gel based electrophoresis technique used in biochemistry, molecular biology, genetics and clinical chemistry for separation of Protein and nucleic acids from a mixture of macromolecules. Agarose is a major component of agar that is used as a matrix in gel baseelectrophoresis. The nucleic acids (DNA and RNA) are separated based on length of fragments while, proteins are separated based on size and/or charge (Kryndushkin, et al. 2003). For separation of bio-molecules the electric field is applied that leads to movement of charged particles via agarose gel matrix and bio-molecul s are separated based on difference in size in the agarose gel matrix (Sambrook and Russel, 2001).

Gel casting with agarose is easy and has very few charged groups, hence found most suitable for nucleic acid separation in most of the biochemistry, molecular biology and biotechnology laboratories. Separated DNA/RNA ma be seen and recorded with stain under UV light. Further DNA fragments can be eluted from agarose gel with an ease for further down applications. The percentage of agarose gel used is in the range of 0.7 - 2. Lower percent of agarose gel is used for long DNA fragments while, higher percent of agarose gel is used for short DNA fragments with suitable running buffer.

Objective: To isolate DNA from plant sample by CTAB method and quantification by spectrophotometer.

Background information:

DNA isolation from different plant tissues viz., leaf, stem, root, flower and seed is a challenging task due to hindrance of variety of variety of biochemical compounds present and further problem increases with diverge plant species. In case of animal tissues the diversity in cellular bio-molecules in different species is much lower compared to plants. Two major phyto bio-molecules that have wide variation between species and pose serious problems in DNA isolation are: polyphenols and polysaccharides. These phyto bio-molecules if contaminated in DNA then may hinder further down applications like PCR, restriction digestion, sequencing, etc.

Several methods have been developed and reported that can remove polyphenols and polysaccharides efficiently during DNA isolation from plants. For this purpose two different chemicals are widely used,the first one is CTAB (cetyl trimethyl ammonium bromide) that is a cationic detergent and helps in removal of polysaccharides and the second one is polyvinylpyrrolidone that helps in removal of polyphenols. Due to above properties the CTAB based DNA extraction buffer is very common for plant DNA extraction.

Significance of different types of chemicals found in CTAB based plant DNA extraction method is explained as under:

Objective: To study peroxidase isozyme variation in rice seedlings by electrophoresis.

Background information

For electrophoresis and PAGE please see practical no. 4. For peroxidase please see practical no. 7.(Page 17-22)

Isozymes (isoenzymes or more generally as multiple forms of enzymes) are the enzymes that are differing in the sequence of amino acids but they work on same substrate or in other words catalyze same biochemical reaction. The isozymes have difference in K_M values and regulatory properties. Isozymes are very useful for proper tuning of metabolic activities and to meet the specific need of particular tissue or developmental stage, e.g. lactate dehydrogenase (LDH). Isozymes are coded by homologous genes that have diversity during evolutionary process. Although, strictly speaking, allozymes represent enzymes from different alleles of the same gene, and isozymes represent enzymes from different genes that process or catalyse the same reaction, the two words are usually used interchangeably.

Objective: To extract and perform enzyme assay of Superoxide dismutase from plant.

Background information:

Superoxide dismutase (SOD, EC 1.15.1.1) is the enzyme that alternately catalyzes the dismutation (or partitioning) of the superoxide (O₂^-) radical into either ordinary molecular oxygen (O₂) or hydrogen peroxide (H₂O₂). Superoxide is produced as a by-product of oxygen metabolism and, if not regulated, may cause cell damage (Hayyan, et al. 2016). Hydrogen peroxide is also responsible for cell damage and may be degraded by other enzymes like catalase. Thus, SOD is an important antioxidant defense in nearly all living cells exposed to oxygen. One exception is Lactobacillus plantarum and related lactobacilli, which use a different mechanism to prevent damage from reactive O₂^-.

Objective: To extract and perform enzyme assay of guaiacol peroxidase from plant.

Background information:

Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:

Objective: To extract and perform enzyme assay of catalase from plant.

Background information

Catalase is a commonly distributed enzyme and present in approximately all organisms exposed to oxygen (such as bacteria, plants and animals). Catalysis of H₂O₂ into H₂O and O₂ is done by catalase enzyme (Goodsell, 2004).