Volume 6 Special Issue 2 2012
Including: Proceedings of Bio-energy from Wastes: Green Chemistry Interventions

ISBN 978-4-907060-00-8

CONTENTS AND ABSTRACTS

Venkatesh Balan, Leonardo da Costa Sousa, Shishir P.S. Chundawat, James Humpula, Bruce E. Dale (USA) Overview to Ammonia Pretreatments for Lignocellulosic Biorefineries (pp 1-11)

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Invited Review: Development of environmentally sustainable and economically viable technologies for plant cell wall deconstruction to fermentable sugars has been impeded due to native plant cell wall recalcitrance to thermochemical and biological based processing. Lower severity alkaline-based pretreatment processes like Ammonia Fiber Expansion (AFEX^TM) can overcome several limitations of traditional pretreatment approaches (e.g., acidic pretreatments) to producing cellulosic biofuels and biochemicals. Here, we give an overview of chemical reactions taking place during alkaline pretreatments including reactions between ammonia and polysaccharides/lignin (e.g., ammonolysis, hydrolysis and Maillard-type reactions). AFEX^TM based pretreatments enhance enzymatic digestibility and fermentability of lignocellulosic biomass through various chemical and ultra-structural modifications within the cell wall. An improved mechanistic understanding of the AFEX^TM process has led to the development of novel alkaline pretreatments that are briefly discussed in this review.

Antje C. Spiess (Germany) Ionic Liquid-Assisted Enzymatic Depolymerisation of Cellulose from Biomass (pp 12-21)

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Invited Review: The recalcitrance of lignocellulose poses a major challenge for its sustainable utilization as source for chemicals, materials and fuels. The capability of some ionic liquids (IL) to dissolve lignocellulose and gain a precipitated amorphous material is exploited within the Cluster of Excellence “Tailor-made fuels from biomass” (www.fuelcenter.rwth-aachen.de) as an alternative pre-treatment for further (bio)-chemical conversion to fuel components. Based on scattered light intensity measurements (BioLector^®, Germany) of cellulose suspended in IL, a number of IL capable of dissolving cellulose could be identified. After precipitation from the IL, enzymatic hydrolysis rates and yields are significantly enhanced (~ 20-fold rate and + 10% yield). These results can be partially transferred to wooden biomass. When retaining e.g. 10% (v/v) ionic liquid content in an aqueous system however, the enzymatic activity of commercial cellulase preparations (Celluclast^®, Novozyme, Denmark) is significantly reduced to between 20 and 30% of its activity in aqueous solution. Ionic strength and viscosity of the IL have been identified as important contributing factors. Interestingly, the enzyme stability was fully maintained. However, the IL interacts differentially for endo- and exo-acting cellulases. The interpretation of these data is facilitated using mathematical models, e.g. those based on population balances (Predici), that allow incorporating both the polymeric nature of the substrate and in case of precipitated cellulose also the particle characteristics of the substrate, i.e. size, crystallinity, and porosity. As a result of the experimental and theoretical studies, improvements for IL-assisted enzymatic hydrolysis should be directed at 1) increasing enzyme activity in presence of IL and 2) tailoring the composition of the enzyme mixture to the resulting cellulosic material using the mathematical models.

Bijaya Ketan Sarangi, Sarita Tiwari, Dinesh Yadav, Ram Avatar Pandey (India) Enhanced Energy Density in Plants: Scope and Prospects (pp 22-31)

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Invited Review: The advances in conversion technologies to make fuels from biomass have become more economically viable, although bioenergy production will primarily depend on biomass availability. However, as biomass availability becomes increasingly restricted, so too does the availability of biomass as a feedstock for bioenergy industry become questionable. Forest resources are the primary source of biomass that can not be further exploited due to a need for environment preservation and sustainable growth. Therefore, R&D strategies to regenerate sufficient feed stock for the bioenergy industry are a priority to make bioenergy production viable. In this context, enhanced biomass production of tree species is a valid R&D proposition which could play key role for foreseeable bioenergy production. This R&D proposition entails an understanding of the regulation of plant radial growth that underlies wood development. Strengthening our knowledge is essential to strategically plan the enhancement of tree biomass production. Phytohormones play a significant role in the regulation of wood development. Auxin is required for cell proliferation and cell differentiation during cambial development. Besides auxin, several other phytohormones, including cytokinin, gibberellin and ethylene, play regulatory roles in the control of cambial activity because of their stimulatory effect on cell division. The potential exploitation of these hormones to enhance biomass production should be explored. This paper outlines R&D approaches to exploit biotechnological tools for manipulation of physiological responses of select woody plants to enhance biomass production.

Madan Junghare, Sanjukta Subudhi, Priyangshu M. Sharma, Ajoy Kumar Mandal, Banwari Lal (India) Mesophilic Hydrogen Production by Clostridium butyricum strain TM-9A, an Alkaline-Tolerant Dark Fermentative Bacterium (pp 32-37)

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Original Research Paper: The present study reports hydrogen production potential by an alkaline-tolerant bacterium Clostridium butyricum strain TM-9A isolated from an estuarine river sediment sample and identified on the basis of 16S rRNA gene sequencing. Different process parameters such as initial pH, temperature and NaCl concentration affected the hydrogen production potential and growth of TM-9A strain in batch dark fermentation experiments. Glucose (10 g L^-1) was used as substrate for an optimization study. TM-9A strain was able to tolerate up to 16 g L^-1 of NaCl. Strain TM-9A produced maximum hydrogen, 57.8 mmol L^-1, at an initial pH 8 under mesophilic conditions, i.e. in the absence of NaCl. Acetic and butyric acid were the major soluble metabolites detected at 12.32 and 11.61 mmol L^-1, respectively. Hydrogen yield was 2.0–2.1 mol H₂/mol glucose. Furthermore, the strain was also evaluated for its ability to utilize different carbohydrate-rich substrates like corn syrup (25.74 mmol L^-1), molasses (23.44 mmol L^-1) and starch (43.29 mmol L^-1), sucrose (31.45 mmol L^-1), and cellulose (4.16 mmol L^-1), respectively for hydrogen production.

Hoysala N. Chanakya, Sreesha Malayil (India) Techno-economic Potential for Value Added Products from Digestion of Urban Solid Wastes and Rural Residues (pp 38-42)

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Original Research Paper: India is urbanizing rapidly accompanied by a gradually increasing rate of municipal solid waste (MSW) production that ranges from 200-500 g/cap/day. The domestic, restaurant, garden and community eatery wastes constitute nearly 90% of the total MSW produced in a city and comprises of >85% decomposable or fermentable components. Decentralized anaerobic digesters are an attractive option that reduces transportation costs and C-footprint but is impeded by difficult to digest components such as banana leaves, stems etc – about 15-20% of the total fermentables of USW. This situation requires simultaneous fermentation of banana leaf/stem along with food wastes. Plug flow approaches allow multi-feed digestion without preprocessing and such an option has been tried out for banana leaf. The feasibility and techno-economic potential of multi-product anaerobic digesters using banana leaf is examined. The operation and function of a 50 kg/d plant (equivalent to a large household plant) is described here. The study quantifies recovery of four value-added products (VAP) sensible to this option and describes the processes required, estimates their value and projects commercial viability – to finally make a case for decentralized, commercially run zero waste option for this type of waste – predominant in south India.

Saumita Banerjee, Ramkrishna Sen, Amruta Morone, Tapan Chakrabarti, Ramavatar Pandey, Sandeep Mudliar (India) Improved Wet Air Oxidation Pretreatment for Enhanced Enzymatic Hydrolysis of Rice Husk for Bioethanol Production (pp 43-45)

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Short Communication: Pretreatment of rice husk by the Alkaline Peroxide Assisted Wet Air Oxidation (APAWAO) approach enhanced the enzymatic convertibility of cellulose in APAWAO-pretreated rice husk. The present work describes the structural changes in rice husk brought about by APAWAO pretreatment by means of Scanning Electron Microscopy (SEM). The SEM images illustrate the extensive loss of biomass integrity following APAWAO pretreatment. X-ray diffraction (XRD) studies indicated the loss of amorphous lignin following APAWAO to be a factor contributing to the enhanced enzymatic digestibility of pre-treated rice husk.

Sridharan Jagadeeswari, Manickam Dakshinamoorthy Balakumaran, Padmanabhan Vidya, Pudupalayam Thangavelu Kalaichelvan, Kanesan Panneer Selvam (India) Utilization of Shrimp and Crab Wastes for the Production of N-Acetylglucosamine by Chitinolytic Soil Streptomyces sp. SJKP9 (pp 46-48)

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Research Note: Shrimp, crab and other crustacean shells are the major wastes of sea food processing industries, possessing a rich amount of protein and oligosaccharides such as chitin and chitosan. In this study, shrimp and crab wastes were utilized to produce the amino sugar compound N-acetylglucosamine using chitinolytic Streptomyces sp. SJKP9 isolated from a sea shore soil sample. The chitin flakes obtained from the sources were digested using crude chitinase enzyme derived from Streptomyces sp. SJKP9 and N-acetylglucosamine was precipitated from a concentrated solution of hydrozylate by adding ethanol. Crab chitin produced more N-acetylglucosamine than shrimp chitin.

Debdulal Banerjee, Subhadip Mahapatra (India) Fungal Tannase: A Journey from Strain Isolation to Enzyme Applications (pp 49-60)

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Invited Review: Tannase is an important enzyme and plays an important role in plant polyphenol degradation as well as conversion to very important pharmaceutical products. Even though this enzyme is widely applied to different food and beverage industries, its industrial production, proper induction and catalysis mechanism still remain limited. This review presents an illustrated revision on fungal tannase. Emphasis had been laid on fungal strains which can produce tannase, different fermentation processes of tannase production, tannase purification, structural and functional characteristics of this enzyme, different enzyme assay techniques and applications of this enzyme. A probable mechanism of tannin degradation by fungus is briefly described.

Rosario Nicoletti, Mario De Stefano (Italy) Penicillium restrictum as an Antagonist of Plant Pathogenic Fungi (pp 61-69)

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Invited Review: The genus Penicillium includes many ubiquitous species which are able to colonize very diverse natural and anthropic contexts as a result of their capacity to adapt to extreme environmental conditions and utilize almost any kind of organic substrate. A number of species are reported to assume agricultural relevance at some extent based on their interactions with cultivated plants and/or other organisms which may sort an effect on the crop outcome. The monoverticillate species Penicillium restrictum is cosmopolitan and mostly regarded as a soil saprotroph. Such a widespread occurrence is reflected in an extensive literature introducing its ecological role and biological properties which are considered and exploited in several biotechnological fields. This review particularly focuses on the antagonistic activity which has been documented against soil-borne plant pathogenic fungi, with special consideration for its recently discovered aptitude to exert mycoparasitism, in view of possible implications for use as an effective biocontrol agent in crop protection.

Shibin Mohanan Nellikunnumal, Arun Chandrashekar (India) Computational Identification of Conserved MicroRNA and their Targets in Coffea canephora by EST Analysis (pp 70-76)

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Original Research Paper: Comparative genomic tools have been successfully used to predict new miRNAs in different plant and animal species using expressed sequence tag (EST) and genome survey sequence (GSS) analysis. In the present study we identified conserved microRNAs in Coffea canephora identified by EST analysis using a new modified comparative genomics method. Use of this method eliminates false positives to a greater extent. Conserved microRNA belonging to 12 families was identified. All identified miRNA were used to search their potential target genes from the SGN (Solanaceace Genome Network) EST database and a total of 42 potential targets were identified for miRNA families from C. canephora. Most of the miRNA targets were transcription factors which appeared to be involved in plant growth, development and stress responses according to the mRNA target information provided by NCBI. The newly identified microRNA can help in better understanding of growth and development Coffea. The improved method can be used for more accurate prediction of microRNA from other plant species with sufficient EST database.

Sarada Ravi, Ranga Rao Ambati, Sandesh B. Kamath, Dayananda Chandrappa, Anila Narayanan, Vikas S. Chauhan, Gokare A. Ravishankar (India) Influence of Different Culture Conditions on Yield of Biomass and Value Added Products in Microalgae (pp 77-85)

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Original Research Paper: Microalgae viz., Botryococcus, Haematococcus and Dunaliella, were studied for their adaptability to different culture conditions. Botryococcus strains of indigenous origin and culture collection centres were studied for their growth, hydrocarbon and lipid profile and for their biomass yields under different bicarbonate concentrations. Growth of Haematococcus was compared in autotrophic (ambient and enriched CO₂ atmosphere) and heterotrophic culture conditions. Influence of ammonium salts, urea and commercial fertilizers as nitrogen source under autotrophic conditions and different amino acids as nitrogen source under heterotrophic culture conditions, was studied on growth and astaxanthin content of Haematococcus. Influence of different salinities was studied on growth and β-Carotene content of Dunaliella in AS100 and De Walnes media. Results indicated that Botryococcus strains varied in their biomass yields (0.25-2 gL^-1), hydrocarbon content (15-60% w/w) and lipid content (15-30%, w/w). An indigenous strain was able to grow at all the tested concentrations of bicarbonate with maximum biomass yield at 0.25 gL^-1. The growth rate of Haematococcus was higher in heterotrophic medium with a cell count of 41-44 × 10⁴ on 5^th day after inoculation. The supplementation of autotrophic medium with 2% CO₂ led to a 4 fold increase in cell count. Use of DAP as nitrogen source showed a 20% increase in yield of encysted biomass. Both DAP and Suphala led to astaxanthin productivity of 46-48 gL^-1. Haematococcus was able to utilize all the amino acids at tested concentrations in heterotrophic culture conditions. Enhanced growth of Dunaliella was observed at 1.0 M NaCl concentration in both the AS 100 and De Walnes media while 2.0M NaCl in De Walnes medium showed higher β-carotene content (45.6 mgg^-1).

Sasidharan Nishanth Kumar, Bala Nambisan, R. Ramya, Chellapan Mohandas (India) Influence of Six Carbon Sources with Yeast Extract on Antimicrobial Metabolite Production by Bacterium Associated with Entomopathogenic Nematode (pp 86-91)

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Original Research Paper: A specific symbiotic Bacillus species isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce a number of bioactive compounds. The present study was conducted to determine the effect of six different carbon sources in combination with yeast extract on the production of antimicrobial substances by Bacillus sp. The yield of crude antimicrobial substances and antimicrobial activity against the test microorganism also differed significantly when the carbon sources in the fermentation media were changed. The highest yield was recorded for maltose plus yeast extract (836 mg/L). The antimicrobial activity was significantly higher in yeast extract plus fructose [Pencillium expansum (46.5 ± 2.12 mm) and Escherichia coli (42.00 mm)] than yeast extract plus other carbon souces used in the study. Antimicrobial activity was significantly reduced in yeast extract plus glucose. HPLC analysis of the crude antimicrobial substances revealed different peaks with different retention times indicating that they produced different compounds. When a carbon source was not included in the fermentation media, the antimicrobial production was substantially reduced to almost eight times. Carbon source in the fermentation medium plays a vital role in the production of antimicrobial substances. Yeast extract and fructose as nitrogen and carbon sources in the fermentation medium produced maximum antimicrobial activity.

Sameen Farha, Emon Chatterjee, Suba G. A. Manuel, Shobha Ananda Reddy, Radha D. Kale (India) Isolation and Characterization of Bioactive Compounds from Fruit Wastes (pp 92-94)

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Short Communication: The current study was aimed at utilizing fruit wastes generated after pectin extraction for assessing their antimicrobial and antioxidant properties. Total soluble proteins (TSP) and heat-stable proteins (HSP) were extracted from wastes of Musa sp., Citrus limetta, Citrullus lanatus, Solanum lycopersicum and Psidium sp. The HSP from S. lycopersicum waste could suppress the growth of Escherichia coli whereas Musa sp.and C. limetta HSP could inhibit the growth of Pseudomonas sp. C. limetta HSP was most effective in suppressing the growth of Fusarium oxysporum relative to the other test samples. No pathogens responded towards the HSP of C. lanatus. High antioxidant activity [Ferric Reducing Antioxidant Power (FRAP)] along with high phenolic levels were observed in Psidium sp. and Musa sp. fruit residues. Adopting appropriate extraction methods for active biomolecules from biodegradable wastes may pave the way for neutriceutical and pharmaceutical applications.

Mithun Raj, Vinayaka Hegde, Muthulekshmi Lajapathy Jeeva, Archana Prathapachandran Vasanthakumari, Pravi Vidyadharan, Vishnu Sukumari Nath, Muthukrishnan Senthil alias Sankar (India) Rapid and Efficient Method for the Extraction of Genomic DNA from Colletotrichum spp. Suitable for PCR Analysis (pp 95-97)

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Techniques Paper: Contemporary approaches for the extraction of genomic DNA from Colletotrichum spp., the filamentous fungal pathogen genera infecting many important crop plants, are often expensive and yield poor quantity and quality of DNA. The high mucilage and polysaccharide content in this fungus add difficulties in genomic DNA isolation, and further downstream applications. We therefore investigated a new and rapid DNA isolation method, which involves inactivation of contaminant proteins by using guanidine hydrochloride/Proteinase K and precipitation of DNA using ice cold isopropanol. This protocol yielded 0.89 ± 0.10 μg DNA mg^-1 of mycelium with purity ranges from 1.75-2.05 as confirmed by A_260/280 spectrophotometric readings. An advantage of this protocol is its compliance even without a refrigerated centrifuge. The new protocol can be successfully used for both mini and maxi preparation of genomic DNA which meet the quality parameters for further downstream processes like PCR-RAPD, AFLP, SSR and ITS amplification of the rDNA-ITS region.