Efficient methane production from agro-industrial residues using anaerobic fungal-rich consortia.

Anaerobic digestion (AD) emerges as a pivotal technique in climate change mitigation, transforming organic materials into biogas, a renewable energy form. This process significantly impacts energy production and waste management, influencing greenhouse gas emissions. Traditional research has largely focused on anaerobic bacteria and methanogens for methane production. However, the potential of anaerobic lignocellulolytic fungi for degrading lignocellulosic biomass remains less explored. In this study, buffalo rumen inocula were enriched and acclimatized to improve lignocellulolytic hydrolysis activity. Two consortia were established: the anaerobic fungi consortium (AFC), selectively enriched for fungi, and the anaerobic lignocellulolytic microbial consortium (ALMC). The consortia were utilized to create five distinct microbial cocktails-AF0, AF20, AF50, AF80, and AF100. These cocktails were formulated based on varying of AFC and ALMC by weights (w/w). Methane production from each cocktail of lignocellulosic biomasses (cassava pulp and oil palm residues) was evaluated. The highest methane yields of CP, EFB, and MFB were obtained at 337, 215, and 54 mL/g VS, respectively. Cocktails containing a mix of anaerobic fungi, hydrolytic bacteria (Sphingobacterium sp.), syntrophic bacteria (Sphaerochaeta sp.), and hydrogenotrophic methanogens produced 2.1-2.6 times higher methane in cassava pulp and 1.1-1.2 times in oil palm empty fruit bunch compared to AF0. All cocktails effectively produced methane from oil palm empty fruit bunch due to its lipid content. However, methane production ceased after 3 days when oil palm mesocarp fiber was used, due to long-chain fatty acid accumulation. Anaerobic fungi consortia showed effective lignocellulosic and starchy biomass degradation without inhibition due to organic acid accumulation. These findings underscore the potential of tailored microbial cocktails for enhancing methane production from diverse lignocellulosic substrates.

Anaerobic microbial cocktail of lignocellulolytic fungi and bacteria with methanogens for boosting methane production from unpretreated rice straw.

Rice straw (RS) has been recognized as a sustainable renewable energy resource for converting into sugars and volatile fatty acids (acetate, propionate, and butyrate) and subsequently to produce biogas. Enhanced production of these intermediates from RS by the different combinations of two consortia was investigated. Anaerobic microbial cocktails of fungi, bacteria, and methanogens were evaluated for performance and stability in the anaerobic digestion of untreated RS. The best-defined anaerobic microbial cocktail for high RS degradation and methane production, consisting of anaerobic bacteria (mainly Proteiniphilum acetatigenes, Pyramidobacter piscolens, and Mesotoga prima) and anaerobic lignocellulolytic/fermentative fungi (uncultured Neocallimastigales, Orpinomyces, Anaeromyces, and Feramyces sp.) at a copy number ratio of 103-105 copies/mL, including hydrogenotrophic and acetoclastic methanogens (Methanosarcina mazei, Methanoculleus marisnigri, Methanofollis liminatans, Methanoculleus bourgensis, and Methanosaeta harundinacea) concentration of 106 copies/mL, was successfully constructed. The system performance was 80% VS (volatile solids) RS degradation, 34 mL/day methane production rate, 318 mL/g VSadded methane yield, and a pH range of 6.90-7.70 within a short time of 14 days. A defined microbial cocktail has been proven as a potential alternative process for lignocellulose hydrolysis and methane production.

Comparison of fermented animal feed and mushroom growth media as two value-added options for waste Cassava pulp management.

Cassava is one of the main processed crops in Thailand, but this generates large amounts (7.3 million tons in 2015) of waste cassava pulp (WCP). The solid WCP is sold directly to farmers or pulp-drying companies at a low cost to reduce the burden of on-site waste storage. Using an integrated direct and environmental cost model, fermented animal feed and mushroom growth media were compared as added-value waste management alternatives for WCP to mitigate environmental problems. Primary and secondary data were collected from the literature, field data, and case studies. Data boundaries were restricted to a gate-to-gate scenario with a receiving capacity of 500 t WCP/d, and based on a new production unit being set up at the starch factory. The total production cost of each WCP utilization option was analyzed from the economic and environmental costs. Fermented animal feed was an economically attractive scenario, giving a higher net present value (NPV), lower investment cost and environmental impact, and a shorter payback period for the 10-year operational period. The selling price of mushrooms was the most sensitive parameter regarding the NPV, while the NPV for the price of fermented animal feed had the highest value in the best-case scenario.

Efficacies of Various Anaerobic Starter Seeds for Biogas Production from Different Types of Wastewater.

Various anaerobic starter seeds from different sources were investigated for their efficacies in treatment of different types of wastewater. Six combinations of starter seeds and wastewaters were selected out of 25 combination batch experiments and operated in semicontinuous reactors. It was noticed that the efficacies of various anaerobic starter seeds for biogas production from different types of wastewater in terms of reactor performance and stability were depended on wastewater characteristics and F/M ratio affecting microbial community and their microbial activities. However, exogenous starter seed can be used across different types of wastewater with or without acclimatization. Four reactors reached the targeted OLR of 2 kg COD/m3·d with high performance and stability except for concentrated rubber wastewater (RBw), even using high active starter seeds of cassava starch (CSs) and palm oil (POs). The toxic compounds in RBw such as ammonia and sulfate might also adversely affect methanogenic activity in CSsRBw and POsRBw reactors. DGGE analysis showed that propionate utilizers, Smithella propionica strain LYP and Syntrophus sp., were detected in all samples. For Archaea domain, methylotrophic, hydrogenotrophic, and acetoclastic methanogens were also detected. Syntrophic relationships were assumed between propionate utilizers and methanogens as acetate/H2 producers and utilizers, respectively.

Enhancement of biogas production from swine manure by a lignocellulolytic microbial consortium.

Anaerobic digestion of lignocellulosic wastes is limited by inefficient hydrolysis of recalcitrant substrates, leading to low biogas yield. In this study, the potential of a lignocellulolytic microbial consortium (LMC) for enhancing biogas production from fibre-rich swine manure (SM) was assessed. Biochemical methane potential assay showed that inoculation of structurally stable LMC to anaerobic digestion led to increase biogas production under mesophilic and thermophilic conditions. The greatest enhancement was observed at 37°C with a LMC/SM ratio of 1.5:1 mg VSS/g VS leading to biogas and methane yields of 355 and 180 ml/g VS(added) respectively, equivalent to 40% and 55% increases compared with the control. The LMC was shown to increase the efficiency of total solid, chemical oxygen demand removal and degradation of cellulose and hemicelluloses (1.87 and 1.65-fold, respectively). The LMC-supplemented process was stable over a 90 d biogas production period. This work demonstrates the potential of LMC for enhancing biogas from lignocellulosic wastes.

Microbial communities and their performances in anaerobic hybrid sludge bed-fixed film reactor for treatment of palm oil mill effluent under various organic pollutant concentrations.

The anaerobic hybrid reactor consisting of sludge and packed zones was operated with organic pollutant loading rates from 6.2 to 8.2 g COD/L day, composed mainly of suspended solids (SS) and oil and grease (O&G) concentrations between 5.2 to 10.2 and 0.9 to 1.9 g/L, respectively. The overall process performance in terms of chemical oxygen demands (COD), SS, and O&G removals was 73, 63, and 56%, respectively. When the organic pollutant concentrations were increased, the resultant methane potentials were higher, and the methane yield increased to 0.30 L CH4/g COD(removed). It was observed these effects on the microbial population and activity in the sludge and packed zones. The eubacterial population and activity in the sludge zone increased to 6.4 × 109 copies rDNA/g VSS and 1.65 g COD/g VSS day, respectively, whereas those in the packed zone were lower. The predominant hydrolytic and fermentative bacteria were Pseudomonas, Clostridium, and Bacteroidetes. In addition, the archaeal population and activity in the packed zone were increased from to 9.1 × 107 copies rDNA/g VSS and 0.34 g COD-CH4/g VSS day, respectively, whereas those in the sludge zone were not much changed. The most represented species of methanogens were the acetoclastic Methanosaeta, the hydrogenotrophic Methanobacterium sp., and the hydrogenotrophic Methanomicrobiaceae.

Decolorization of molasses melanoidins and palm oil mill effluent phenolic compounds by fermentative lactic acid bacteria.

Lactobacillus plantarum SF5.6 is one of the lactic acid bacteria (LAB) that has the highest ability of molasses melanoidin (MM) decolorization among the 2114 strains of LAB. The strains were isolated from spoilage, pickle fruit and vegetable, soil and sludge from the wastewater treatment system by using technical step of enrichment, primary screening and secondary screening. This LAB strain SF5.6 was identified by 16S rDNA analysis and carbohydrate fermentation (API 50 CH). The top five LAB strains having high MM decolorization (> 55%), namely TBSF5.8-1, TBSF2.1-1, TBSF2.1, FF4A and SF5.6 were selected to determine the optimal condition. It was found that the temperature at 30 degrees C under facultative conditions in GPY-MM medium (0.5% glucose, 0.1% peptone, 0.1% yeast extract, 0.1% sodium acetate, 0.05% MgSO4 and 0.005% MnCl2 in MM solution at pH 6) giving a high microbial growth and MM decolorization for all five strains. It was noticed that the decolorization of MM by LAB strains might be cell growth associated. L. plantarum SF5.6 grew rapidly within one day while the other strains took 2-3 days. This L. plantarum SF5.6 could rapidly decolorize MM to 60.91% without any lag phase, and it also had the ability to remove 34.00% phenolic compounds and 15.88% color from treated palm oil mill effluent.

Effect of recirculation rate on methane production and SEBAR system performance using active stage digester.

A project was undertaken to examine the feasibility of treating organic wastes from Thai fruit and vegetable markets using the sequential batch anaerobic digester (SEBAR) approach. A key feature of the SEBAR system is the regular interchanging, or recirculation, of portions of leachate between each freshly filled digester and a support digester to which it is coupled until it is ready to operate independently. Leachate transfer from this support digester to the fresh waste digester provides additional alkalinity to help counteract the effects of early high acid release rates; it also helps build a balanced microbial population in the fresh waste digester. To optimize the leachate recirculation process, the effect of varying the quantities of leachate interchanged between freshly filled waste digesters and the still highly active support digesters to which they were coupled was studied. It was found that increasing the recirculation rate accelerated the onset of both waste degradation and methane production. The increasing of recirculation rate from 10% to 20% and 10% to 30% could reduce the SEBAR cycle period by approximately 7% and 22% without significant reduction in the amount of methane obtained from the systems. The methane yields were 0.0063, 0.0068 and 0.0077 l g(-1) VS added in the NEW digester per day using leachate recirculation rates of 10%, 20% and 30%, respectively. This finding has potentially important practical and economic implications for those using the SEBAR system to add value to market waste.

Neural-fuzzy control system application for monitoring process response and control of anaerobic hybrid reactor in wastewater treatment and biogas production.

Based on the developed neural-fuzzy control system for anaerobic hybrid reactor (AHR) in wastewater treatment and biogas production, the neural network with backpropagation algorithm for prediction of the variables pH, alkalinity (Alk) and total volatile acids (TVA) at present day time t was used as input data for the fuzzy logic to calculate the influent feed flow rate that was applied to control and monitor the process response at different operations in the initial, overload influent feeding and the recovery phases. In all three phases, this neural-fuzzy control system showed great potential to control AHR in high stability and performance and quick response. Although in the overloading operation phase II with two fold calculating influent flow rate together with a two fold organic loading rate (OLR), this control system had rapid response and was sensitive to the intended overload. When the influent feeding rate was followed by the calculation of control system in the initial operation phase I and the recovery operation phase III, it was found that the neural-fuzzy control system application was capable of controlling the AHR in a good manner with the pH close to 7, TVA/Alk < 0.4 and COD removal > 80% with biogas and methane yields at 0.45 and 0.30 m3/kg COD removed.

Combination effect of pH and acetate on enzymatic cellulose hydrolysis.

The productivity and efficiency of cellulase are significant in cellulose hydrolysis. With the accumulation of volatile fatty acids (VFAs), the pH value in anaerobic digestion system is reduced. Therefore, this study will find out how the pH and the amount of acetate influence the enzymatic hydrolysis of cellulose. The effects of pH and acetate on cellulase produced from Bacillus coagulans were studied at various pH 5-8, and acetate concentrations (0-60 mmol/L). A batch kinetic model for enzymatic cellulose hydrolysis was constructed from experimental data and performed. The base hypothesis was as follows: the rates of enzymatic cellulose hydrolysis rely on pH and acetate concentration. The results showed that the suitable pH range for cellulase production and cellulose hydrolysis (represents efficiency of cellulase) was 2.6-7.5, and 5.3-8.3, respectively. Moreover, acetate in the culture medium had an effect on cellulase production (K(I) = 49.50 mmol/L, n = 1.7) less than cellulose hydrolysis (K(I) = 37.85 mmol/L, n = 2.0). The results indicated that both the pH of suspension and acidogenic products influence the enzymatic hydrolysis of cellulose in an anaerobic environment. To enhance the cellulose hydrolysis rate, the accumulated acetate concentration should be lower than 25 mmol/L, and pH should be maintained at 7.

Contributions of available substrates and activities of trophic microbial community to methanogenesis in vegetative and reproductive rice rhizospheric soil.

Potential of methane production and trophic microbial activities at rhizospheric soil during rice cv. Supanbunri 1 cultivation were determined by laboratory anaerobic diluents vials. The methane production was higher from rhizospheric than non-rhizospheric soil, with the noticeable peaks during reproductive phase (RP) than vegetative phase (VP). Glucose, ethanol and acetate were the dominant available substrates found in rhizospheric soil during methane production at both phases. The predominance activities of trophic microbial consortium in methanogenesis, namely fermentative bacteria (FB), acetogenic bacteria (AGB), acetate utilizing bacteria (AB) and acetoclastic methanogens (AM) were also determined. At RP, these microbial groups were enhanced in the higher of methane production than VP. This correlates with our finding that methane production was greater at the rhizospheric soil with the noticeable peaks during RP (1,150 +/- 60 nmol g dw(-1) d(-1)) compared with VP (510 +/- 30 nmol g dw(-1) d(-1)). The high number of AM showed the abundant (1.1x10(4) cell g dw(-1)) with its high activity at RP, compared to the less activity with AM number at VP (9.8x10(2) cell g dw(-1)). Levels of AM are low in the total microbial population, being less than 1% of AB. These evidences revealed that the microbial consortium of these two phases were different.

Anaerobic degradation kinetics of reactive dye with different carbon sources.

This study aims to investigate the anaerobic degradation kinetics of reactive dye, C.I. Reactive Red 141 (Evercion Red H-E7B) by partially granulated anaerobic mixed culture using three carbon sources, namely modified starch (MS), polyvinyl alcohol (PVA) and acrylic size (AS) during batch incubation. There is a first-order kinetics reaction in the decolorization processes using MS and PVA as carbon sources, while a zero-order kinetics relationship describes the decolorization process for the AS carbon source. The k values and color removal rate of decolorization with MS carbon source was higher than those of PVA and AS carbon sources. This is because the MS carbon source was well degraded in comparison to AS and PVA, respectively This study also found dye reduction could be enhanced through the addition of MS as a carbon source. The decolorization rates increased with decrease in dye concentrations of RR 141. In contrast, the decolorization rates increased with increase in COD concentration.

Molecular monitoring of microbial population dynamics during operational periods of anaerobic hybrid reactor treating cassava starch wastewater.

This study characterized the microbial community and population dynamics in an anaerobic hybrid reactor (AHR) treating cassava starch wastewater. Methanogens and nonmethanogens were followed during the start-up and operation of the reactor, and linked to operational and performance data. Biomass samples taken from the sludge bed and packed bed zones of the AHR at intervals throughout the operational period were examined by 16S rRNA fluorescence in situ hybridization (FISH). The start-up seed and the reactor biomass were sampled during the feeding of the wastewater with a chemical oxygen demand (COD) value of 8 g L(-1) and a hydraulic retention time (HRT) of 8 days. These samples were characterized by the predominance of cells with long-rod morphology similar to Methanosaeta spp. Following a sharp operational change, accomplished by increasing the COD concentration of the organic influent from 8 to 10 g L(-1) and reducing the HRT from 8 to 5 days, there was a doubling of the organic loading rate, a reduction of the COD removal efficiency, as well as decreased methane content in the biogas and an accumulation of total volatile acids in the reactor. Moreover, this operational change resulted in a significant population shift from long-rod Methanosaeta-like cells to tetrad-forming Methanosarcina-like cells. The distributions of microbial populations involved in different zones of the AHR were determined. The results showed that nonmethanogens became the predominant population in both sludge and the packed bed zone. However, the percentage of methanogens in the packed bed zone was higher than that in the sludge bed zone. This higher percentage of methanogens was likely caused by the fact that the packed bed zone provided a suitable environmental condition with an appropriate nutrient availability for methanogen growth.

Factors affecting hydrogen production from cassava wastewater by a co-culture of anaerobic sludge and Rhodospirillum rubrum.

Series of batch experiments were used to investigate the effects of environmental factors, i.e., total nitrogen and total phosphorus concentrations, initial pH, illumination pattern and stirring conditions on hydrogen production from cassava wastewater by a co-culture of anaerobic sludge and Rhodospirillum rubrum. The maximum of the hydrogen yield of 150.46 and 340.19 mL g-COD(-1) was obtained at the total nitrogen and total phosphorus concentrations of 0.2 and 0.04 M, respectively. An effect of initial pH was investigated at COD:N:P ratio of 100:10:1. Results indicated that an optimum initial pH for hydrogen production was pH 7 with a high hydrogen yield of 158.78 mL g-COD(-1) was obtained. No significantly different (p < 0.05) in the effect of illumination pattern (24 h of light and 12 h dark/light cycle) on hydrogen production were observed under continuous-illumination and periodic-illumination with hydrogen yield of 131.84 and 126.92 mL g-COD(-1), respectively. Therefore, a periodic-illumination was applicable in hydrogen fermentation due to its cost-effective. Hydrogen fermentation with a stirring at 100 rpm provided more effective hydrogen production (164.83 mL g-COD(-1)) than static-fermentation (93.93 mL g-COD(-1)). The major soluble products from hydrogen fermentation were acetic and butyric acids, in the ranges of 28.33-48.30 and 35.23-66.07%, respectively, confirming an ability of a co-culture to produce hydrogen from cassava wastewater.

Use of an alternative Archaea-specific probe for methanogen detection.

An alternative 16S rRNA-targeted oligonucleotide probe specific for Archaea was developed and used for detection of methanogens in anaerobic reactors. The designed probe was checked for its specificity by computer-aided comparative sequence analysis. For in situ application, optimal stringency conditions were adjusted by performing whole cell hybridization using target and nontarget organisms. Anaerobic sludge samples were examined by in situ hybridization for methanogenic populations. The relative abundance of methanogens was monitored with epifluorescence microscopy. Individual cells could be visualized with strong fluorescence signals after hybridization with the newly developed probe.

Use of fluorochrome-labeled rRNA targeted oligonucleotide probe and tyramide signal amplification to improve sensitivity of fluorescence in situ hybridization.

A tyramide signal amplification (TSA) system was used in combination with a conventional fluorochrome-labeled 16S rRNA oligonucleotide probe to increase the sensitivity of fluorescence in situ hybridization. TSA was performed after hybridization resulted in a low fluorescence signal intensity. In contrast to the horseradish peroxidase-tyramide signal amplification (HRP-TSA) system and biotin-tyramide signal amplification (biotin-TSA) system, no additional expensive probe labeling was required. A whole cell hybridization technique was used to compare the fluorescence signal obtained using a monolabeled probe with that obtained using the TSA system. The fluorescence signal of the probe obtained using the TSA system was much higher than that obtained using the monolabeled probe. The technique was successfully applied to the in situ detection of microbial communities in anaerobic sludge. It was demonstrated that TSA resulted in an increased in sensitivity, as the fluorescence signal intensity was much higher than that obtained using a conventional probe.

Nylon fibers as supporting media in anaerobic hybrid reactors: it's effects on system's performance and microbial distribution.

The performances of three anaerobic hybrid reactors with various nylon fiber densities per packed bed volume (33, 22, and 11 kg/m(3) in R1, R2, and R3, respectively) as supporting media were evaluated through their ability to remove organic compounds in cassava starch wastewater. In addition, the distributions of non-methanogenic and methanogenic population in the reactors were investigated. During a 6-month operation, the organic loading rate was increased in stepwise from 0.5 to 4.0 kg COD/m3/day and the hydraulic retention time (HRT) shortened to 5.4 days. The COD removal efficiency was more favorable in R1 (87%) and R2 (84%) than in R3 (70%). The total biomass in the reactors with greater nylon fiber densities was also higher and increased from 20.4 to 67.3 g VSS and to 57.5 g VSS in R1 and R2, respectively. When the HRT was further shortened to 3 days, however, the efficiency of both reactors demonstrated a declining trend and reached 74% in R1 and 61% in R2. The distribution of microbial populations involved in the reactors was determined using the Most Probable Number technique. The result showed the lowest number of methanogens in R3 which correlated well to its relatively low efficiency. The number of non-methanogens in all reactors was, nonetheless, comparable. By shortening the HRT to 3 days, the methanogenic population in R2 diminished in both attached and suspended biomass whereas a slight reduction was detected only in the attached biomass of R1.