Successive process for efficient biovalorization of Brewers' spent grain to lignocellulolytic enzymes and lactic acid production through simultaneous saccharification and fermentation.

This study aimed to increase the value of brewers' spent grain (BSG) by using it as feedstock to produce lignocellulolytic enzymes and lactic acid (LA). Twenty-two fungal strains were screened for lignocellulolytic enzyme production from BSG. Among them, Trichoderma sp. showed the highest cellulase activity (35.84 ± 0.27 U/g-BSG) and considerably high activities of xylanase (599.61 ± 23.09 U/g-BSG) and ß-glucosidase (16.97 ± 0.77 U/g-BSG) under successive solid-state and submerged fermentation. The processes were successfully scaled up in a bioreactor. The enzyme cocktail was recovered and characterized. The maximum cellulase and xylanase activities were found at pH 5.0 and 50 °C, and the activities were highly stable at pH 4-8 and 30-50 °C. The enzyme cocktail was applied in simultaneous saccharification and fermentation of acid-pretreated BSG for LA production. The maximum LA obtained was 59.3 ± 1.0 g/L. This study has shown the efficient biovalorization of BSG, and this approach may also be applicable to other agro-industrial wastes.

Chitosan-coated oleaginous microalgae-fungal pellets for improved bioremediation of non-sterile secondary effluent and application in carbon dioxide sequestration in bubble column photobioreactors.

Oleaginous microalga Scenedesmus sp. SPP was rapidly immobilized in oleaginous fungal pellets by their opposite-surface-charges. Microalgae-fungal (MF) pellets were more effective in bioremediation of non-sterile secondary effluent than mono-culture. The optimal hydraulic retention time for dual bioremediation in semi-continuous mode was 72 h. The MF pellets coated with 0.4 %-chitosan improved removal efficiencies of COD, total nitrogen (TN), and total phosphorus (TP) up to 96.2±0.0 %, 88.2±2.8 % and 71.5±0.7 %, respectively, likely because of better cell retention and more nutrient adsorption and assimilation. Dual bioremediation by coated MF pellets was also successfully scaled up in 30-L bubble-column photobioreactors with improved COD, TN, and TP removal efficiencies of 98.5±0.0 %, 90.2±0.0 % and 79.5±2.1 %, respectively. This system also effectively removed CO2 from simulated flue gas at 71.2±0.4 % and produced biomass with high lipid content. These results highlight the effectiveness of bio-immobilization by fungal pellets; chitosan coating; and their practical applications in bioremediation and CO2 sequestration.

Isolation and screening of microorganisms for high yield of succinic acid production.

This study involves the isolation of succinic acid (SA)-producing microorganisms from different samples, including the rumen, sludge, soil, and wastewater. For primary screening, 29 isolates exhibited a zone of clearance around the colony, indicating acid production. For secondary screening using thin-layer chromatography, only two isolates symbolized SA production according to their Rf values. These two isolates were further identified as Bacillus velezensis and Enterococcus gallinarum by phylogenetic analysis using the neighbor-joining method. The high SA concentrations of 50.2 and 66.9 g/L were produced by B. velezensis and E. gallinarum with an SA yield of 0.836 and 1.12 g/g glucose, respectively. The high SA concentration from these newly isolated strains was achieved with a low formation of unwanted acids compared with those from Actinobacillus succinogenes ATCC 55618. Moreover, E. gallinarum was cultured in palm oil mill wastewater (POMW) and molasses, which were cheap substrates. The high SA production of 73.9 g/L with low other acids (the ratio of SA to total acids = 0.917) was achieved using POMW and molasses (80:20) as substrates.

Purification and characterization of a highly-stable fungal xylanase from Aspergillus tubingensis cultivated on palm wastes through combined solid-state and submerged fermentation.

Fungal xylanase was produced from lignocellulosic palm wastes through combined solid-state fermentation (SSF) and submerged fermentation (SmF) by Aspergillus tubingensis TSIP9 in a helical-impeller equipped bioreactor. The combined SSF-SmF promoted the xylanase production by 15 and 70% higher than SSF and SmF, respectively. Sequential purification yielded 7.4-fold purified xylanase with 9.07% recovery. The maximum activities of crude and purified xylanase were observed at the same pH of 5.0 and the same temperature of 50 °C while purified xylanase is more active and highly stable at a wider pH range of 3-8 and temperature of 30-60 °C. The half-life of purified xylanase at various temperatures was also much improved by 2-8 folds compared to crude xylanase. Michaelis-Menten constants, Vmax and Km, for purified xylanase are 2,602.8 U/mg and 32.4 mg/mL, respectively. Purified xylanase activity was most enhanced with Ca2+ followed by Zn2+ and Fe2+ at 10 mM while significantly inhibited by Co2+, Cu2+, Pb2+, and Ag+. This study has shown the effectiveness of combined SSF-SmF for xylanase production and superior properties of purified xylanase for industrial processes.

Microbial fuel cells with Photosynthetic-Cathodic chamber in vertical cascade for integrated Bioelectricity, biodiesel feedstock production and wastewater treatment.

This study aimed to develop efficient microbial fuel cells (MFCs) for integrated bioelectricity, biodiesel feedstock production and wastewater treatment. Among wastewaters tested, MFC fed with anaerobic digester effluent from rubber industry gave the maximum power density (55.43 ± 1.08 W/m3) and simultaneously removed COD, nitrogen and phosphorus (by 72.4 ± 0.9%, 40.5 ± 0.8% and 24.4 ± 1.5%, respectively). 16S rRNA gene analysis revealed that dominant microbial communities were: Firmicutes (43.68%), Bacteroidetes (25.41%) and Chloroflexi (15.02%), which mostly contributed to bioelectricity generation. After optimizing organic loading rate, photosynthetic oleaginous microalgae were applied in cathodic chamber in order to increase oxygen availability, secondarily treat anodic chamber effluent and produce lipids as biodiesel feedstocks. Four MFCs with photosynthetic-cathodic chamber connected in vertical cascade could improve power density up to 116.9 ± 15.5 W/m3, sequentially treat wastewater, and also produce microalgal biomass (465 ± 10 g/m3) with high lipid content (38.17 ± 0.01%). These strategies may greatly contribute to sustainable development of integrated bioenergy generation and environment.

Consolidated bioprocesses for efficient bioconversion of palm biomass wastes into biodiesel feedstocks by oleaginous fungi and yeasts.

Consolidated bioprocesses for bioconversion of lignocellulosic biomass into biodiesel feedstocks were developed. Palm empty fruit bunch (EFB) was biologically pretreated coupling with fungal lipid production (121.4 ± 2.7 mg/g-EFB) by lignocellulolytic oleaginous fungi prior to lipid production by oleaginous yeasts. In subsequent separate hydrolysis and fermentation (SHF) of fungal pretreated EFB (FPEFB), the oleaginous yeast with the maximum lipid yield of 37.0 ± 0.1 mg/g-FPEFB was screened. While a higher lipid yield of 47.9 ± 1.5 mg/g-FPEFB was achieved in simultaneous saccharification and fermentation (SSF) with less enzyme requirement. Fed-batch SSF of non-sterile FPEFB was proven as a practical and efficient strategy to increase lipid yield up to 53.4 ± 0.5 mg/g-FPEFB. Total lipid yield by both fungi and yeast was 165.0 ± 4.4 mg/g-EFB. Interestingly, the consolidated bioprocesses of enzyme and lipid production also achieved comparable total lipid yield of 149.3 ± 6.6 mg/g-EFB. These strategies may contribute greatly to cost-effective and sustainable bioconversion of lignocellulosic biomass into biodiesel feedstocks.

Valorization of palm biomass wastes for biodiesel feedstock and clean solid biofuel through non-sterile repeated solid-state fermentation.

Palm biomass wastes are currently considered as promising solid biofuels. However, their high potassium content leads to formation of slag in combustion chambers and causes frequent power-plant shutdowns for maintenance. Therefore, this study aimed to develop a low-cost practical biological pretreatment for these wastes. Oleaginous fungi Aspergillus tubingensis TSIP9, which originates from palm wastes, was used to pretreat biomass wastes and simultaneously produce oils through non-sterile solid state fermentation (SoSF). The operating conditions were optimized through response surface methodology. The fungi could grow and produce oils with good biodiesel fuel properties. After SoSF, potassium content in biomass wastes was reduced by 90% and cellulose content increased to >57%, making it suitable as clean solid biofuel. Repeated-SoSF with 90% substrate replacement was highly effective in continuously pretreating biomass wastes and producing fungal oils. This study demonstrates the cost-effective and environmentally friendly process for production of clean renewable energy through zero-waste strategy.

Anaerobic digestion of hydrothermally-pretreated lignocellulosic biomass: Influence of pretreatment temperatures, inhibitors and soluble organics on methane yield.

Anaerobic digestion (AD) of lignocellulosic biomass has received significant attention for bioenergy production in recent years. However, hydrolysis is a rate-limiting in AD of such feedstock. In this study, effects of hydrothermal pretreatment of Napier grass, a model lignocellulosic biomass, on methane yield were examined through series of batch and semi-continuous studies. In batch studies, the highest methane yield of 248.2 ±â€¯5.5 NmL CH4/g volatile solids (VS)added was obtained from the biomass pretreated at 175 °C, which was 35% higher than that from the unpretreated biomass. The biomass pretreated at 200 °C resulted in formation of 5-hydroxymethylfurfural and furfural, which significantly inhibited methanogenesis. In semi-continuous studies, digester fed with the biomass pretreated at 200 °C at organic loading rate (OLR) of 4 g VS/L.d resulted in digester failure. Thus, OLRsoluble/OLRtotal ratio <200 is proposed as an operating criterion for effective operation of digester fed with pretreated biomass slurry.

Immobilized oleaginous microalgae as effective two-phase purify unit for biogas and anaerobic digester effluent coupling with lipid production.

Oleaginous microalga Scenedesmus sp. was immobilized in alginate-gel beads and applied as two-phase purify unit for biogas and anaerobic digester effluent from palm oil mill. Optimal microalgal cell concentration and bead volume ratio were 106 cells mL-1 and 25% v/v, respectively. The use of 20% effluent and light intensity at 128 µmol·proton·m-2 s-1 most promoted CO2 removal by immobilized microalgae and achieved the maximum CO2 removal rate of 4.63 kg-CO2 day-1 m-3. This process upgraded methane content in biogas (>95%) and completely remove nitrogen and phosphorus in the effluent. After process operation, 2.98 g L-1 microalgal biomass with 35.92% lipid content were recovered by simple sieving method. Microalgal lipids are composed of C16-C18 (>98%) with prospect high cetane number and short ignition delay time. This study has shown the promising biorefinery concept which is effective not only in CO2 fixation, biogas upgrading and pollutant removal but also cost-effective production of microalgae-based biofuel.

Enhancing Electricity Generation Using a Laccase-Based Microbial Fuel Cell with Yeast Galactomyces reessii on the Cathode.

The fungi associated with termites secrete enzymes such as laccase (multi-copper oxidase) that can degrade extracellular wood matrix. Laccase uses molecular oxygen as an electron acceptor to catalyze the degradation of organic compounds. Owing to its ability to transfer electrons from the cathodic electrode to molecular oxygen, laccase has the potential to be a biocatalyst on the surface of the cathodic electrode of a microbial fuel cell (MFC). In this study, a two-chamber MFC using the laccase-producing fungus Galactomyces reessii was investigated. The fungus cultured on coconut coir was placed in the cathode chamber, while an anaerobic microbial community was maintained in the anode chamber fed by industrial rubber wastewater and supplemented by sulfate and a pH buffer. The laccase-based biocathode MFC (lbMFC) produced the maximum open circuit voltage of 250 mV, output voltage of 145 mV (with a 1,000 Ω resistor), power density of 59 mW/m2, and current density of 278 mA/m2, and a 70% increase in half-cell potential. This study demonstrated the capability of laccase-producing yeast Galactomyces reessii as a biocatalyst on the cathode of the two-chamber lbMFC.

Co-production of functional exopolysaccharides and lactic acid by Lactobacillus kefiranofaciens originated from fermented milk, kefir.

Kefiran is a functional exopolysaccharide produced by Lactobacillus kefiranofaciens originated from kefir, traditional fermented milk in the Caucasian Mountains, Russia. Kefiran is attractive as thickeners, stabilizers, emulsifiers, gelling agents and also has antimicrobial and antitumor activity. However, the production costs of kefiran are still high mainly due to high cost of carbon and nitrogen sources. This study aimed to produce kefiran and its co-product, lactic acid, from low-cost industrial byproducts. Among the sources tested, whey lactose (at 2% sugar concentration) and spent yeast cells hydrolysate (at 6 g-nitrogen/L) gave the highest kefiran of 480 ± 21 mg/L along with lactic acid of 20.1 ± 0.2 g/L. The combination of these two sources and initial pH were optimized through Response Surface Methodology. With the optimized medium, L. kefiranofaciens produced more kefiran and lactic acid up to 635 ± 7 mg/L and 32.9 ± 0.7 g/L, respectively. When the pH was controlled to alleviate the inhibition from acidic pH, L. kefiranofaciens could consume all sugars and produced kefiran and lactic acid up to 1693 ± 29 mg/L and 87.49 ± 0.23 g/L, respectively. Moreover, the fed-batch fermentation with intermittent adding of whey lactose improved kefiran and lactic acid productions up to 2514 ± 93 mg/L and 135 ± 1.75 g/L, respectively. These results indicate the promising approach to economically produce kefiran and lactic acid from low-cost nutrient sources.

Removal of hydrogen sulfide generated during anaerobic treatment of sulfate-laden wastewater using biochar: Evaluation of efficiency and mechanisms.

Removal of hydrogen sulfide (H2S) from biogas was investigated in a biochar column integrated with a bench-scale continuous-stirred tank reactor (CSTR) treating sulfate-laden wastewater. Synthetic wastewater containing sulfate concentrations of 200-2000mg SO42-/L was used as substrate, and the CSTR was operated at an organic loading rate of 1.5g chemical oxygen demand (COD)/L·day and a hydraulic retention time (HRT) of 20days. The biochar was able to remove about 98.0 (±1.2)% of H2S for the ranges of concentrations from 105-1020ppmv, especially at high moisture content (80-85%). Very high H2S adsorption capacity (up to 273.2±1.9mg H2S/g) of biochar is expected to enhance the H2S oxidation into S0 and sulfate. These findings bring a potentially novel application of sulfur-rich biochar as a source of sulfur, an essential but often deficient micro-nutrient in soils.

Mitigation of carbon dioxide by oleaginous microalgae for lipids and pigments production: Effect of light illumination and carbon dioxide feeding strategies.

Oleaginous microalgae Nannochloropsis sp. was selected as potential strain for CO2 mitigation into lipids and pigments. The synergistic effects of light intensity and photoperiod were evaluated to provide the adequate light energy for this strain. The saturation light intensity was 60µmol·photon·m(-2)s(-1). With full illumination, the biomass obtained was 0.850±0.16g·L(-1) with a lipid content of 44.7±1.2%. The pigments content increased with increasing light energy supply. Three main operating factors including initial cell concentration, CO2 content and gas flow rate were optimized through Response Surface Methodology. The feedings with low CO2 content at high gas flow rate gave the maximum biomass but with low lipid content. After optimization, the biomass and lipid production were increased up to 1.30±0.103g·L(-1) and 0.515±0.010g·L(-1), respectively. The CO2 fixation rate was as high as 0.729±0.04g·L(-1)d(-1). The fatty acids of Nannochloropsis sp. lipids were mainly C16-C18 indicating its potential use as biodiesel feedstocks.

Optimization of flocculation efficiency of lipid-rich marine Chlorella sp. biomass and evaluation of its composition in different cultivation modes.

This study aimed to optimize flocculation efficiency of lipid-rich marine Chlorella sp. biomass and evaluate its composition in different cultivation modes. Among three flocculants including Al(3+), Mg(2+) and Ca(2+) tested, Al(3+) was most effective for harvesting microalgal biomass. Four important parameters for flocculation were optimized through response surface methodology. The maximum flocculation efficiency in photoautotrophic culture was achieved at pH 10, flocculation time of 15 min, Al(3+) concentration of 2.22 mM and microalgal cells of 0.47 g/L. The flocculation in mixotrophic culture required lower amount of Al(3+) (0.74 mM) than that in photoautotrophic and heterotrophic cultures (2.22 mM). The biomass harvested from mixotrophic culture contained lipid at the highest content of 42.08 ± 0.58% followed by photoautotrophic (32.08 ± 3.88%) and heterotrophic (30.42 ± 1.13%) cultures. The lipid-extracted microalgal biomass residues (LMBRs) contained protein as high as 38-44% and several minerals showing their potential use as animal feed and their carbohydrate content were 16-29%.

Upflow bio-filter circuit (UBFC): biocatalyst microbial fuel cell (MFC) configuration and application to biodiesel wastewater treatment.

A biodiesel wastewater treatment technology was investigated for neutral alkalinity and COD removal by microbial fuel cell. An upflow bio-filter circuit (UBFC), a kind of biocatalyst MFC was renovated and reinvented. The developed system was combined with a pre-fermented (PF) and an influent adjusted (IA) procedure. The optimal conditions were operated with an organic loading rate (OLR) of 30.0 g COD/L-day, hydraulic retention time (HRT) of 1.04 day, maintained at pH level 6.5-7.5 and aerated at 2.0 L/min. An external resistance of circuit was set at 10 kΩ. The purposed process could improve the quality of the raw wastewater and obtained high efficiency of COD removal of 15.0 g COD/L-day. Moreover, the cost of UBFC system was only US$1775.7/m3 and the total power consumption was 0.152 kW/kg treated COD. The overall advantages of this invention are suitable for biodiesel wastewater treatment.

Removal of H2S in down-flow GAC biofiltration using sulfide oxidizing bacteria from concentrated latex wastewater.

A biofiltration system with sulfur oxidizing bacteria immobilized on granular activated carbon (GAC) as packing materials had a good potential when used to eliminate H(2)S. The sulfur oxidizing bacteria were stimulated from concentrated latex wastewater with sulfur supplement under aerobic condition. Afterward, it was immobilized on GAC to test the performance of cell-immobilized GAC biofilter. In this study, the effect of inlet H(2)S concentration, H(2)S gas flow rate, air gas flow rate and long-term operation on the H(2)S removal efficiency was investigated. In addition, the comparative performance of sulfide oxidizing bacterium immobilized on GAC (biofilter A) and GAC without cell immobilization (biofilter B) systems was studied. It was found that the efficiency of the H(2)S removal was more than 98% even at high concentrations (200-4000 ppm) and the maximum elimination capacity was about 125 g H(2)S/m(3)of GAC/h in the biofilter A. However, the H(2)S flow rate of 15-35 l/h into both biofilters had little influence on the efficiency of H(2)S removal. Moreover, an air flow rate of 5.86 l/h gave complete removal of H(2)S (100%) in biofilter A. During the long-term operation, the complete H(2)S removal was achieved after 3-days operation in biofilter A and remained stable up to 60-days.

Effect of nitrate on the performance of single chamber air cathode microbial fuel cells.

The effect of nitrate on the performance of a single chamber air cathode MFC system and the denitrification activity in the system were investigated. The maximum voltage output was not affected by 8.0mM nitrate in the medium solution at higher external resistance (270-1000Omega), but affected at lower resistance (150Omega) possibly due to the low organic carbon availability. The Coulombic efficiency was greatly affected by the nitrate concentration possibly due to the competition between the electricity generation and denitrification processes. Over 84-90% of nitrate (0.8-8.0mM) was removed from the single chamber MFCs in less than 8h in the first batch. After 4-month operation, over 85% of nitrate (8.0mM) was removed in 1h after the MFC was continuously fed with a medium solution containing nitrate. Only a small amount of nitrite (<0.01mM) was detected during the denitrification process. The similar denitrification activity observed at different external resistances (1000 and 270Omega) and open circuit mode indicates that the denitrification was not significantly affected by the electricity generation process. No electricity was generated when the MFC fed with 8.0mM nitrate was moved to a glove box (no oxygen), indicating that the bacteria on the cathode did not involve in accepting electrons from the circuit to reduce the nitrate. Denaturing Gradient Gel Electrophoresis (DGGE) profiles demonstrate a similar bacterial community composition on the electrodes and in the solution but with different dominant species.