Search

A Martelella endophytica (M. endophytica) strain YC6887 was previously isolated from the roots of a halophyte, Rosa rugosa, which was sequenced and characterized. The genomic and proteomic analysis showed a carbohydrate-degrading enzyme, endoglucanase Cel5A which was further characterized. The protein analysis revealed that this endoglucanase belongs to glycosidic hydrolase family 5 (GH5) with catalytic domain. This gene encodes 349-residue polypeptide and shows closest similarity with cellulases of other Martelella species. The protein was purified to homogeneity and shown that it was a 39 kDa protein. The purified recombinant Cel5A endoglucanase exhibited maximum activity at 50 °C and pH 4.5. The enzyme was salt tolerant and retained more than 50% residual activity up to 15% NaCl. The homology model structure of Cel5A displayed that it is stable and compact protein structure consisting of eleven α-helical structures and eight β-sheets. According to the predicted ligand binding site after superimposition with Pseudomonas stutzeri endoglucanase Cel5A (PDB ID: 4LX4), it consisted of five amino acid Asn157, Tyr116, Glu158, Glu270 and Trp303 that may be the expected active site of Cel5A from YC6887. This presented that our strain M. endophytica YC6887 that produces cellulase partially degrade the insoluble polysaccharides into reducing sugars.

This study aims to determine UV-B resistance and to investigate computational analysis and antioxidant potential of methoxy-flavones of Micromonospora aurantiaca TMC-15 isolated from Thal Desert, Pakistan. The cellular extract was purified through solid phase extraction and UV-Vis spectrum analysis indicated absorption peaks at λmax 250 nm, 343 nm, and 380 nm that revealed the presence of methoxy-flavones named eupatilin and 5-hydroxyauranetin. The flavones were evaluated for their antioxidant as well as protein and lipid peroxidation inhibition potential using di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium (DPPH), 2,4-dinitrophenyl hydrazine (DNPH), and thiobarbituric acid reactive substances (TBARS) assays, respectively. The methoxy-flavones were further studied for their docking affinity and interaction dynamics to determine their structural and energetic properties at atomic level. The antioxidant potential, protein and lipid oxidation inhibition and DNA damage preventive abilities were correlated as predicted by computational analysis. The eupatilin and 5-hydroxyauranetin binding potential to their targeted proteins 1N8Q and 1OG5 is -4.1 and -7.5 kcal/mol, respectively. Moreover, the eupatiline and 5-hydroxyauranetin complexes illustrate van der Waals contacts and strong hydrogen bonds to their respective enzymes target. Both in-vitro studies and computational analysis results revealed that methoxy-flavones of Micromonospora aurantiaca TMC-15 can be used against radiation mediated oxidative damages due to its kosmotrophic nature. The demonstration of good antioxidant activities not only protect DNA but also protein and lipid oxidation and therefore could be a good candidate in radio-protective drugs and as sunscreen due to its kosmotropic nature.

Biodiesel is one of the best alternative to depleting fossil fuels for transport sector. However, biodiesel production via lipase mediated transesterification has limitation of high costing microbial enzymes. In order to overcome this limitation, a process of sequential treatment of oil industry wastewater using isolated lipolytic bacterial strains and biodiesel production from non-edible plant oils was studied. In this study, efficient lipase producing bacteria were isolated and evaluated for production of biodiesel from mustard, soybean, jatropha and taramira oils utilizing methanol for the transesterification of oils and bioremediation. Selected strains were then identified, using 16s rRNA sequencing. Further, Bacillus subtilis strain Q1 KX712301 was optimized for biodiesel production from non-edible taramira oil via Plackett–Burman and central composite design. Highest volumetric yield of biodiesel obtained was 102% at optimized parameters. Finally, a sequential bioremediation of vegetable oil contaminated wastewater and then microbial production of biodiesel from non-edible taramira oil was carried out using efficient lipase producer B. subtilis strain Q1 at optimized conditions. During sequential process, complete chemical oxigen demand reduction of oil containing wastewater and theoretical volumetric yield of biodiesel was achieved. Gas chromatography/mass spectrometry chromatogram revealed that the total fatty acid methyl ester content of the produced biodiesel was >98% which is in accordance with the biodiesel quality standards specified by both ASTM and EU-14103.

Hydrolytic bacteria are essential for the degradation of lignocellulose to produce biogas and organic fertilizers. In this study, sheep manure was used as substrate, and sheep manure slurry, yak rumen fluid and slurry from a biogas reactor (SBR) were used as inocula in single-stage anaerobic digestion. The SBR and rumen fluid inocula increased biogas production by 23% and 43%, respectively, when compared to solely sheep manure in the single-stage anaerobic digestion. The two-stage anaerobic digestion, with yak rumen fluid as inoculum in the hydrolytic reactor, increased the biogas production by 59, 86, and 58% compared with the control. Microbial analysis of the effluent revealed that yak rumen fluid contained hydrolytic bacteria such as Proteiniphilum, Jeotgalibaca, Fermentimonas, and Atopostipes to enhance the degradation of sheep manure and increase biogas production. It was concluded that yak rumen fluid, rich in hydrolytic bacteria, increases the degradability of sheep manure and improves production of volatile fatt acids and biogas.

Most of the presently known β-glucosidases are sensitive to end-product inhibition by glucose, restricting their potential use in many industrial applications. Identification of novel glucose tolerant β-glucosidase can prove a pivotal solution to eliminate end-product inhibition and enhance the overall lignocellulosic saccharification process. In this study, a novel gene encoding β-glucosidase BglNB11 of 1405bp was identified in the genome of Saccharomonospora sp. NB11 and was successfully cloned and heterologously expressed in E. coli BL21 (DE3).The presence of conserved amino acids; NEPW and TENG indicated that BglNB11 belonged to GH1 β-glucosidases. The recombinant enzyme was purified using a Ni-NTA column, with the molecular mass of 51 kDa, using SDS-PAGE analysis. BglNB11 showed optimum activity at 40 °C and pH 7 and did not require any tested co-factors for activation. The kinetic values, Km, Vmax, kcat, and kcat/Km of purified enzyme were 0.4037 mM, 5735.8 μmol/min/mg, 5042.16 s-1 and 12487.71 s-1 mM-1, respectively. The enzyme was not inhibited by glucose to a concentration of 4 M but was slightly stimulated in the presence of glucose. Molecular docking of BglNB11 with glucose suggested that the relative binding position of glucose in the active site channel might be responsible for modulating end product tolerance and stimulation. β-glucosidase from BglNB11 is an excellent enzyme with high catalytic efficiency and enhanced glucose tolerance compared to many known glucose tolerant β-glucosidases. These unique properties of BglNB11 make it a prime candidate to be utilized in many biotechnological applications.

During in situ biogas up-gradation by supplying hydrogen from an external source and enrichment of hydrogenotrophic methanogens, high pressure of H2 negatively affects hydrolytic and fermentative activities. To overcome this problem, the present study aimed to enrich the hydrogenotrophic methanogens by optimization of various parameters associated with gas recirculation along-with hydrogen supply from the external source. Due to recirculation of gases and supplied hydrogen, methane generation was two-fold higher in the optimal condition than in conventional anaerobic digestion, with the highest methane content of 99%. Additionally, the hydrogenotrophic methanogens were enriched, with a decrease in acetoclastic methanogens and an increase in Bathyarchaeia population, which utilizes H2 and CO2 to produce acetate and lactate as end products. The study concludes that recirculation increases methane production by converting H2 and CO2 into methane and enhances the degradation of organic matter left over undigested in the hydrolytic reactor.

Lignin is a major byproduct of pulp and paper industries, which is resistant to depolymerization due to its heterogeneous structure. The enzymes peroxidases can be utilized as potent bio-catalysts to degrade lignin. In the current study, an Efeb gene of 1251bp encoding DyP-type peroxidase from Bacillus sp. strain BL5 (DyPBL5) was amplified, cloned into a pET-28a (+) vector and expressed in Escherichia coli BL21 (DE3) cells. A 46 kDa protein of DyPBL5 was purified through ion-exchange chromatography. Purified DyPBL5 was active at wide temperature (25-50 °C) and pH (3.0-8.0) range with optimum activity at 35 °C and pH 5.0. Effects of different chemicals on DyPBL5 were determined. The enzyme activity was strongly inhibited by SDS, DDT and β-mercaptoethanol, whereas stimulated in the presence of organic solvents such as methanol and ethanol. The kinetic parameters were determined and Km, Vmax and Kcat values were 1.06 mM, 519.75 μmol/min/mg and 395 S 1, respectively. Docking of DyPBL5 with ABTS revealed that, Asn 244, Arg 339, Asp 383 and Thr 389 are putative amino acids, taking part in the oxidation of ABTS. The recombinant DyPBL5 resulted in the reduction of lignin contents up to 26.04 %. The SEM and FT-IR analysis of test samples gave some indications about degradation of lignin by DyPBL5. Various low molecular weight lignin degradation products were detected by analyzing the samples through gas chromatography mass spectrometry. High catalytic efficiency and lignin degradation rate make DyPBL5 an ideal bio-catalyst for remediation of lignin-contaminated sites.

Waste activated sludge generated from wastewater treatment plants makes an abundant source of biomass. Its effective utilization through anaerobic digestion (AD) requires pretreatment to disintegrate the sludge matrix and increase organic matter availability. In this study, dewatered waste activated sludge (DWAS) was subjected to alkaline, photocatalytic, and alkaline-photocatalytic pretreatment for its disintegration and subsequent methane production using different concentrations of sodium hydroxide and titania nanoparticles. Individual pretreatment resulted in maximum disintegration degree (DDsCOD) of 11.3 and 5.2% at 0.8% NaOH and 0.6 gTiO2/L, respectively. Alkaline-photocatalytic pretreatment yielded 37% DDsCOD at 0.8% NaOH–0.4 g/L TiO2. As compared to control, AD at 0.4% NaOH and 0.5 g/L TiO2 pretreatments yielded maximum methane, which was 50.4 and 32.6% higher. Similarly, alkaline-photocatalytic pretreatment at 0.4% NaOH-0.5 g/L TiO2 yielded methane as 462 N mL/g VS, which was 71.1% higher. Modified Gompertz model fitted the methane yield data well.

A cellulase-producing bacterium, designated as strain AK9, was isolated from a hot spring of Tatta Pani, Azad Kashmir, Pakistan. The bacterium was identified as Bacillus amyloliquefaciens through 16S rRNA sequencing. Cellulase from strain AK9 was able to liberate glucose from soluble cellulose and carboxymethyl cellulose (CMC). Enzyme was purified through size exclusion chromatography and a single band of ∼47 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified with recovery of 35.5%, 3.6-fold purity with specific activity of 31 U mg−1. The purified cellulase retained its activity over a wide range of temperature (50–70 °C) and pH (3–7) with maximum stability at 60 °C and pH 5.0. The activity inhibited by ethylenediaminetetraacetic acid (EDTA), suggested that it was metalloenzyme. Diethyl pyrocarbonate (DEPC) and β-mercaptoethanol significantly inhibited cellulase activity that revealed the essentiality of histidine residues and disulfide bonds for its catalytic function. It was stable in non-ionic surfactants, in the presence of various metal ions, and in water-insoluble organic solvents. Approximately 9.1% of reducing sugar was released after enzymatic saccharification of DAP-pretreated agro-residue, compared to a very low percentage by autohydrolysis treatment. Hence, it is concluded that cellulase from B. amyloliquefaciens AK9 can potentially be used in bioconversion of lignocellulosic biomass to fermentable sugars.

A process for the production of high bioethanol titre was established through fed-batch and simultaneous saccharification and fermentation (FB-SSF) of wild, non-edible cassava Manihot glaziovii. FB-SSF allowed fermentation of up to 390g/L of starch-derived glucose achieving high bioethanol concentration of up to 190g/L (24% v/v) with yields of around 94% of the theoretical value. The wild cassava M. glaziovii starch is hydrolysable with a low dosage of amylolytic enzymes (0.1-0.15% v/w, Termamyl® and AMG®). The Automatic Gas Potential Test System (AMPTS) was adapted to yeast ethanol fermentation and demonstrated to be an accurate, reliable and flexible device for studying the kinetics of yeast in SSF and FB-SSF. The bioethanol derived stoichiometrically from the CO2 registered in the AMPTS software correlated positively with samples analysed by HPLC (R(2)=0.99).

The feasibility of anaerobic treatment of methanol condensate from pulp and paper mill in UASB reactor was investigated and compared with the anaerobic treatment of methanol. The UASB reactor treating methanol condensate was operated for 480 days with minimum problems of overload. COD removal from methanol condensate and methanol under normal operating conditions ranged from 84-86% to 86-98%, respectively. Under optimal conditions (OLR=5.0 g COD L(-1)day(-1), COD(influent)=11.40 g L(-1)) a methane yield of 0.29 NL CH(4) per g COD(removed) (at standard temperature and pressure) was achieved during the treatment of methanol condensate. The recovery time of the microorganisms after several overloads was decreasing each time probably due to the adaptation to methanol condensate. These findings indicate that methanol condensate can be efficiently treated in a UASB reactor with the benefit of biogas production. As a bonus effect of the treatment, much of the smell of the feed was eliminated.

The objective of this study was to characterise and evaluate a wild inedible cassava species, Manihot glaziovii as feedstock for bioenergy production. Tubers obtained from 3 different areas in Tanzania were characterised and evaluated for bioethanol and biogas production. These bioenergy carriers were produced both separately and sequentially and their energy values evaluated based on these two approaches. Composition analysis demonstrated that M. glaziovii is a suitable feedstock for both bioethanol and biogas production. Starch content ranged from 77% to 81%, structural carbohydrates 3-16%, total crude protein ranged from 2% to 8%. Yeast fermentation achieved ethanol concentration of up to 85g/L at a fermentation efficiency of 89%. The fuel energy of the bioethanol and methane from flour-peels mix ranged from 5 to 13 and 11 to 14MJ/kgVS, respectively. Co-production of bioethanol and biogas in which the peels were added to the fermentation residue prior to anaerobic digestion produced maximum fuel energy yield of (15-23MJ/kgVS).

A multi-channel analyzer was used to evaluate biogas potential of sugarcane bagasse (SCB). The Automatic Methane Potential Test System contained fifteen parallel reactors and the same number of gas flow meters attached to the acquisition system. The set of reactors - gas flow meters gave reproducible results during anaerobic digestion of chemically defined carbon source and the units were used to evaluate the biomethane potential of SCB after different pretreatments, such as treatment with water, acid, acid followed by enzymatic treatment and acid followed by treatment with inactive enzymes. Combined pretreatment with 2% sulphuric acid and enzymatic hydrolysis (3.5% enzymes) resulted in conversion of 79% to monomeric sugars present in SCB. SCB treated with acid followed by enzymatic hydrolysis achieved the methane yield of 200NL per kg VS(added). Enzymatic saccharification of acid pretreated SCB resulted in increase of methane yield by 16±5% compared to that from acid treated SCB. (Less)

The recently isolated anaerobic bacterium Caloramator boliviensis with an optimum growth temperature of 60 °C can efficiently convert hexoses and pentoses into ethanol. When fermentations of pure sugars and a pentose-rich sugarcane bagasse hydrolysate were carried out in a packed bed reactor with immobilized cells of C. boliviensis, more than 98% of substrates were converted. Ethanol yields of 0.40-0.46 g/g of sugar were obtained when sugarcane bagasse hydrolysate was fermented. These features reveal interesting properties of C. boliviensis in producing ethanol from a renewable feedstock.