Bioethanol (a renewable resource), blended with gasoline, is used as liquid transportation fuel worldwide and produced from either starch or lignocellulose. Local production and use of bioethanol supports local economies, decreases country's carbon footprint and promotes self-sufficiency. The latter is especially important for bio-resource-rich land-locked countries like Nepal that are seeking alternative transportation fuels and technologies to produce them. In that regard, in the present study, we have used two highly efficient ethanol producing yeast strains, viz., Saccharomyces cerevisiae (CDBT2) and Wickerhamomyces anomalous (CDBT7), in an electrochemical cell to enhance ethanol production. Ethanol production by CDBT2 (anodic chamber) and CDBT7 (cathodic chamber) control cultures, using 5% glucose as substrate, were 12.6 ± 0.42 and 10.1 ± 0.17 mg·mL⁻¹ respectively. These cultures in the electrochemical cell, when externally supplied with 4V, the ethanol production was enhanced by 19.8 ± 0.50% and 23.7 ± 0.51%, respectively, as compared to the control cultures. On the other hand, co-culturing of those two yeast strains in both electrode compartments resulted only 3.96 ± 0.83% enhancement in ethanol production. Immobilization of CDBT7 in the graphite cathode resulted in lower enhancement of ethanol production (5.30 ± 0.82%), less than free cell culture of CDBT7. CDBT2 and CDBT7 when cultured in platinum nano particle coated platinum anode and neutral red-coated graphite cathode, respectively, ethanol production was substantially enhanced (52.8 ± 0.44%). The above experiments when repeated using lignocellulosic biomass hydrolysate (reducing sugar content was 3.3%) as substrate, resulted in even better enhancement in ethanol production (61.5 ± 0.12%) as compared to glucose. The results concluded that CDBT2 and CDBT7 yeast strains produced ethanol efficiently from both glucose and lignocellulosic biomass hydrolysate. Ethanol production was enhanced in the presence of low levels of externally applied voltage. Ethanol production was further enhanced with the better electron transport provision i.e., when neutral red was deposited on cathode and fine platinum nanoparticles were coated on the platinum anode.

Microorganisms producing laccases may be used for the pretreatment of lignocellulosic biomass to recover fermentable sugar. Very few fungi and other microbes growing in high altitudes have been tested for this purpose. As part of this study, we have collected soil samples from different parts of the Kathmandu Valley and the Rautah at district of Nepal (1600 to 2303 m above sea level) and successfully cultured 53 different isolates of microorganisms. Among the 53 isolates obtained 30 were Actinomycetes, 20 were Streptomycetes, and three were fungi). These isolates were tested for laccase expression using guaiacol, tannic acid, and 1-naphthol as substrates. Twelve of the 53 isolates tested positive for the expression of laccase. Among the laccase- positive isolates, a fungal species designated as CDBT-F-G1was found to produce high levels of laccase. This isolate was identified as Pestalotiopsis species based on 18S rRNA sequencing. Pestalotiopsis spp. CDBT-F-G1 isolate grows efficiently in PDB media containing 1% Kraft lignin at pH 5 and 30 °C and secretes 20 ± 2 U/mL laccase in culture medium. Further optimization of growth conditions reveled that addition of (i) metal salts, e.g., 1 mM magnesium sulfate (51 ± 25 U/mL); (ii) agitation of cultures at 200 rpm (51 ± 9U/mL); (iii) surfactants, e.g., 0.75 mM Tween 80 (54 ± 14 U/mL); (iv) 40% dissolved O₂ (57 ± 2 U/mL) and inducers, e.g., 1 mM gallic acid (69 ± 11 U/mL), further promote laccase production by Pestalotiopsis spp. CDBT-F-G1 isolate. On the other hand, 0.1 mM cysteine inhibited laccase production. The secretory laccase obtained from fermentation broth of CDBT-F-G1 was partially purified by ammonium sulfate (13-fold purification with specific activity 26,200 U/mg) and acetone (14-fold purification with specific activity 31,700 U/mg) precipitation methods. The enzyme has an approximate molecular mass of 43 kDa, pH and temperature optima werepH6 and 60 °C, respectively. V_max and K_m were 100 μmol/min and 0.10 mM, respectively, with ABTS as the substrate. Given the above characteristics, we believe Pestalotiopsis spp. CDBT-F-G1 strain native to high altitudes of Nepal could be used to pretreat lignocellulosic biomass to efficiently recover fermentable sugars.

Fungal consortium of a cellulolytic isolate F1 (Aspergillus niger) and/or ligninocellulolytic isolate F2 (Ganoderma sessile) along with Saccharomyces cerevisiae (CDBT2) were used for integrated approach of saccharification, fermentation and electrofermentation of Saccharum spontaneum biomass to produce ethanol. Different consortium sets of these isolates were used to observe fermentation efficiency. All the analysis were done using crude as well as hot water pretreated S. spontaneum powder to compare the ethanol production efficiency. The ethanol yield was found to be higher in crude biomass compared to hot water pretreated S. spontaneum biomass. At 28°C, 80 rpm and 5.0 pH, simultaneous culture with combination of G. sessile and CDBT2 produced higher amount of ethanol (57 mg/g crude biomass) followed by combination of G. sessile, A. niger and CDBT2 (44 ± 0.18 mg/g crude biomass). However, the amount of phenol and furfural generated were very low in both cases. In addition, electro-fermentation enhanced ethanol production by 12% and 18% with a combination culture of G. sessile, CDBT2 and G. sessile, A. niger, CDBT2 respectively. This method of lignocellulosic ethanol production by simultaneous saccharification and electrofermentation can be developed as an alternative approach for cost effective production of bioethanol.

Microbial fuel cell (MFC) is an alternative way for household organic waste treatment as it produces sustainable clean energy. Chemical parameters of the cauliflower leaf waste before treatment were 143.5 mg/g COD, 0.90 mg/g ammoniacal nitrogen, 67.8 mg/g phosphorous, 214.8 mg/g total reducing sugar and 0.53 mg/g soluble reducing sugar. After eight days of treatment in MFC at the optimised condition of multiwalled carbon nanotubules (MWCNT)’ coated graphite as anode and potassium ferricyanide added phosphate buffer as catholyte with an external resistance of 1000 Ω showed a reduction in COD, ammoniacal nitrogen, phosphorous and total reducing sugar by 24.7, 76.9, 22.5 and 53.4%, respectively, along with a maximum power density of 10.1 W/m³. Scanning electron microscopy (SEM) showed bacterial adherence in the graphite electrode and its molecular characterisation using 16S rRNA sequencing confirmed as Bacillus sps.

In view of crude oil prices, and its environmental issues, utilization of sustainable renewable alternative energies such as biofuels is rapidly progressing in many countries. The increasing global energy demand and depleting fossils fuels sources has led to search alternative clean and renewable fuels. One of the best alternatives to the gasoline is lignocellulosic bioethanol. Recent researches on lignocellulosic bioethanol focuses on advancement of pretreatment techniques for improved sugar yields and decreased inhibitors production. Pretreatment technique with no or less use of chemicals and cost effectiveness is the main purpose of most of the researches. Biological pretreatment techniques produce less fermentation inhibitors than chemical pretreatments. In order to cope with fermentation inhibitors different strategies can be adopted during pretreatment processes. In the course of time, advancements in production process over separate hydrolysis and fermentation have been introduced. Simultaneous saccharification and co/fermentation; and consolidated bioprocessing for bioethanol production are gaining popularity among researchers. Int. J. Appl. Sci. Biotechnol. Vol 10(1): 1-11.

Biogas has long been used as a household cooking fuel in many tropical counties, and it has the potential to be a significant energy source beyond household cooking fuel. In this study, we describe the use of low electrical energy input in an anaerobic digestion process using a microbial electrochemical cell (MEC) to promote methane content in biogas at 18, 28, and 37 °C. Although the maximum amount of biogas production was at 37 °C (25 cm³), biogas could be effectively produced at lower temperatures, i.e., 18 (13 cm³) and 28 °C (19 cm³), with an external 2 V power input. The biogas production of 13 cm³ obtained at 18 °C was ~65-fold higher than the biogas produced without an external power supply (0.2 cm³). This was further enhanced by 23% using carbon-nanotubes-treated (CNT) graphite electrodes. This suggests that the MEC can be operated at as low as 18 °C and still produce significant amounts of biogas. The share of CH₄ in biogas produced in the controls was 30%, whereas the biogas produced in an MEC had 80% CH₄. The MEC effectively reduced COD to 42%, whereas it consumed 98% of reducing sugars. Accordingly, it is a suitable method for waste/manure treatment. Molecular characterization using 16s rRNA sequencing confirmed the presence of methanogenic bacteria, viz., Serratia liquefaciens and Zoballella taiwanensis, in the inoculum used for the fermentation. Consistent with recent studies, we believe that electromethanogenesis will play a significant role in the production of value-added products and improve the management of waste by converting it to energy.