Sudip Kumar Rakshit

Developments in industrially important thermostable enzymes: a review

Cellular components of thermophilic organisms (enzymes, proteins and nucleic acids) are also thermostable. Apart from high temperature they are also known to withstand denaturants of extremely acidic and alkaline conditions. Thermostable enzymes are highly specific and thus have considerable potential for many industrial applications. The use of such enzymes in maximising reactions accomplished in the food and paper industry, detergents, drugs, toxic wastes removal and drilling for oil is being studied extensively. The enzymes can be produced from the thermophiles through either optimised fermentation of the microorganisms or cloning of fast-growing mesophiles by recombinant DNA technology. In this review, the source microorganisms and properties of thermostable starch hydrolysing amylases, xylanases, cellulases, chitinases, proteases, lipases and DNA polymerases are discussed. The industrial needs for such specific thermostable enzyme and improvements required to maximize their application in the future are also suggested.

Comparison of some new pretreatment methods for second generation bioethanol production from wheat straw and water hyacinth

Pretreatment of lignocellulosic residues like water hyacinth (WH) and wheat straw (WS) using crude glycerol (CG) and ionic liquids (IL) pretreatment was evaluated and compared with conventional dilute acid pretreatment (DAT) in terms of enzymatic hydrolysis yield and fermentation yield of pretreated samples. In the case of WS, 1-butyl-3-methylimidazolium acetate pretreatment was found to be the best method. The hydrolysis yields of glucose and total reducing sugars were 2.1 and 3.3 times respectively higher by IL pretreatment than DAT, while it was 1.4 and 1.9 times respectively higher with CG pretreatment. For WH sample, CG pretreatment was as effective as DAT and more effective than IL pretreatment regarding hydrolysis yield. The fermentation inhibition was not noticeable with both types of pretreatment methods and feedstocks. Besides, CG pretreatment was found as effective as pure glycerol pretreatment for both feedstocks. This opens up an attractive economic route for the utilization of CG.

Ultrasound Pretreatment of Cassava Chip Slurry to Enhance Sugar Release for Subsequent Ethanol Production

The use of ultrasound pretreatment to enhance liquefaction and saccharification of cassava chips was investigated. Cassava chip slurry samples were subjected to sonication for 10–40 s at three power levels of low (2 W/mL), medium (5 W/mL), and high (8 W/mL). The samples were simultaneously exposed to enzymes to convert starch into glucose. The cassava particle size declined nearly 40‐fold following ultrasonic pretreatment at high power input. Scanning electron micrographs of both unsonicated (control) and sonicated samples showed disruption of fibrous material in cassava chips but did not affect the granular structure of starch. Reducing sugar release improved in direct proportion to the power input and sonication time. The reducing sugar increase was as much as 180% with respect to the control groups. The slurry samples with enzyme addition during sonication resulted in better reducing sugar release than the samples with enzyme addition after sonication. The heat generated during sonication below starch gelatinization temperature apparently had no effect on the reducing sugar release. The reducing sugar yield and energy efficiency of ultrasound pretreated samples increased with total solids (TS) contents. The highest reducing sugar yield of 22 g/100 g of sample and efficiency of 323% were obtained for cassava slurry with 25% TS at high power. The reducing sugar yield at the completion of reaction (R∞) were over twofold higher compared to the control groups. The integration of ultrasound into a cassava‐based ethanol plant may significantly improve the overall ethanol yield. Biotechnol. Bioeng. 2008;101: 487–496.

Monoclonal antibody production using a new supermacroporous cryogel bioreactor

A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long‐term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551–564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (∼20 days), the hybridoma cell line consumed 0.75 mM day−1 glucose, produced 2.48 mM day−1 lactic acid, and produced 6.5 μg mL−1 day−1 mAb during the exponential phase. The mAb concentration reached 130 μg mL−1 after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L−1, which was three times greater than the mAb yield obtained from T‐flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL‐1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice.

Identification and Characterization of a Cellulase-Encoding Gene from the Buffalo Rumen Metagenomic Library

Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose–degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.

Forest biorefinery: Potential of poplar phytochemicals as value-added co-products

The global forestry industry after experiencing a market downturn during the past decade has now aimed its vision towards the integrated biorefinery. New business models and strategies are constantly being explored to re-invent the global wood and pulp/paper industry through sustainable resource exploitation. The goal is to produce diversified, innovative and revenue generating product lines using on-site bioresources (wood and tree residues). The most popular product lines are generally produced from wood fibers (biofuels, pulp/paper, biomaterials, and bio/chemicals). However, the bark and other tree residues like foliage that constitute forest wastes, still remain largely an underexploited resource from which extractives and phytochemicals can be harnessed as by-products (biopharmaceuticals, food additives and nutraceuticals, biopesticides, cosmetics). Commercially, Populus (poplar) tree species including hybrid varieties are cultivated as a fast growing bioenergy crop, but can also be utilized to produce bio-based chemicals. This review identifies and underlines the potential of natural products (phytochemicals) from Populus species that could lead to new business ventures in biorefineries and contribute to the bioeconomy. In brief, this review highlights the importance of by-products/co-products in forest industries, methods that can be employed to extract and purify poplar phytochemicals, the potential pharmaceutical and other uses of >160 phytochemicals identified from poplar species - their chemical structures, properties and bioactivities, the challenges and limitations of utilizing poplar phytochemicals, and potential commercial opportunities. Finally, the overall discussion and conclusion are made considering the recent biotechnological advances in phytochemical research to indicate the areas for future commercial applications from poplar tree species.

Antinutritional factors and in vitro protein digestibility of improved haricot bean (Phaseolus vulgaris L.) varieties grown in Ethiopia

The antinutrient (raffinose oligosaccharides, tannins, phytic acid and trypsin inhibitors) composition and in vitro protein digestibility of eight improved varieties of Phaseolus vulgaris grown in Ethiopia were determined. Stachyose was the predominant α-galactosides in all haricot bean samples. Raffinose was also present in significant quantities but verbascose, glucose and fructose were not detected at all in the samples. The concentrations observed for the protein digestibility and antinutritional factors, varied significantly (P<0.05) between varieties investigated in this study. Mean values for protein digestibility ranged from 80.66% (in Roba variety) to 65.64% (in Beshbesh variety). Mean values for raffinose, stachyose, sucrose, trypsin inhibitors, tannins and phytic acid were 3.14 mg/g, 14.86 mg/g, 24.22 mg/g, 20.68 TUI×103/g, 17.44 mg, catechin equivalents/g and 20.54 mg/g respectively. Statistical analyses of data revealed that antinutritional factors and protein digestibility were influenced by...The antinutrient (raffinose oligosaccharides, tannins, phytic acid and trypsin inhibitors) composition and in vitro protein digestibility of eight improved varieties of Phaseolus vulgaris grown in Ethiopia were determined. Stachyose was the predominant α-galactosides in all haricot bean samples. Raffinose was also present in significant quantities but verbascose, glucose and fructose were not detected at all in the samples. The concentrations observed for the protein digestibility and antinutritional factors, varied significantly (P<0.05) between varieties investigated in this study. Mean values for protein digestibility ranged from 80.66% (in Roba variety) to 65.64% (in Beshbesh variety). Mean values for raffinose, stachyose, sucrose, trypsin inhibitors, tannins and phytic acid were 3.14 mg/g, 14.86 mg/g, 24.22 mg/g, 20.68 TUI×103/g, 17.44 mg, catechin equivalents/g and 20.54 mg/g respectively. Statistical analyses of data revealed that antinutritional factors and protein digestibility were influenced by variety (genotype). Relationships between antinutritional factors and protein digestibility were also observed. The possibility of selecting varieties to be used for large-scale cultivation in Ethiopia on the basis of these data is discussed. Among the improved varieties studied, Roba, Redwolaita, Mexican and Awash were found to be the best food and export type of haricot beans in the Ethiopian context, because of their higher protein digestibility, lower antinutrtional factors and other beneficial nutritional parameters. Roba variety can be used by local food processors for the production of value-added bean-based products especially to combat the problem of protein energy malnutrition and related diseases which are very common in developing countries.

Effect of different pretreatments on delignification pattern and enzymatic hydrolysability of miscanthus, oil palm biomass and typha grass

Cumulative pretreatments methods were evaluated for delignification ability and enzymatic digestibility using miscanthus (M×G), empty palm fruit bunch (EFB) and typha grass as feedstocks. Despite their close chemical composition, the three feedstocks unveiled quite different behavior under the same condition of pretreatment. Characterization of ethanol organosol lignins extracted from the three feedstocks by (13)C NMR and FTIR revealed information concerning S/G/H ratios which was important to rationalize the differences among the feedstock behavior. The S/G/H ratios for MxG, EFB and typha, were established to levels of ~52/44/4, ~68/30/2 and ~46/27/27 respectively. The xylans hydrolytic susceptibility were a major cause of difference in behavior of feedstock during the pretreatment process. The influence of the presence of naphthol during autohydrolysis on the delignification ability was studied. A good relationship was observed between S+H/G ratio and the scavenging effect of naphthol.

Three-Dimensional Culture for Monoclonal Antibody Production by Hybridoma Cells Immobilized in Macroporous Gel Particles

Cell proliferation and long‐term production of monoclonal antibody IgG2b by M2139 hybridoma cells immobilized in macroporous gel particles (MGPs) in packed‐bed reactor were studied for a period of 60 days. The MGPs were made of supermacroporous gels produced in frozen conditions from crosslinked polyacrylamide and modified with gelatin which were housed in special plastic carriers (7 × 9 mm2). Cells were trapped in the interior part of MGPs by attaching to the void space of the gel matrix as three‐dimensional (3D) cultivation using gelatin as a substrate layer. Optimizing productivity by hybridoma cell relies on understanding regulation of antibody production. In this study, the behavior of M2139 cells in two‐dimensional cultures on multiwell plate surfaces was also investigated. The effect of three different medium such as basal medium Dulbeccos modified Eagles medium (D‐MEM) containing L‐glutamine or L‐glutamine + 2 mM α‐ketoglutarate or L‐alanyl‐glutamine (GlutaMAX™) was studied prior to its use in 3D cultivation. The kinetics of cell growth in basal medium containing L‐glutamine + α‐ketoglutarate was similar to cells grown on GlutaMAX containing medium, whereas D‐MEM containing L‐glutamine showed lower productivity. With the maximal viable cell density (6.85 × 106 cells mL−1) and highest specific mAb production rate (3.9 μg mL−1 10−4 viable cell day−1), D‐MEM‐GlutaMAX was further selected for 3D cultivation. Cells in MGPs were able to grow and secrete antibody for 30 days in packed‐bed batch reactor, before a fresh medium reservoir was replaced. After being supplied with fresh medium, cells again showed continuous growth for another 30 days with mAb production efficiency of 50%. These results demonstrate that MGPs can be used efficiently as supporting carrier for long‐term monoclonal antibody production.

Biogas production by encased bacteria in synthetic membranes: protective effects in toxic media and high loading rates.

A bioreactor including encased digesting bacteria for biogas production was developed, and its performance in toxic media and under high organic loading rates (OLRs) was examined and compared with traditional digestion reactors. The bacteria (3 g) were encased and sealed in 3×6 cm2 PVDF (polyvinylidene fluoride) membranes with a pore size of 0.1 μm, and then several sachets were placed in the reactors. They were then examined in toxic medium containing up to 3% limonene as a model inhibitor in batch reactors, and OLRs of up to 20 g COD/L.day in semi-continuous digestions. The free and encased cells with an identical total bacterial concentration of 9 g in a medium containing 2% limonene produced at most 6.56 and 23.06 mL biogas per day, respectively. In addition, the digestion with free cells completely failed at an OLR of 7.5 g COD/L.day, while the encased cells were still fully active with a loading of 15 g COD/L.day.