High Volatile Fatty Acids Production Research Articles

Enhancement of volatile fatty acids to extremely high content in fermentation of food waste: Optimization of conditions, microbial functional genes, and mechanisms

The engineering application of volatile fatty acids (VFA) production from food waste (FW) can significantly enhance resource utilization. Enhancing VFA production is crucial for advancing this engineering application. This study presented a economically-feasible method to achieve high VFA production from FW: Conducting fermentation at pH 9 and 37 ℃ with addition of 20 % anaerobic sludge significantly increased the conversion of FW to VFAs (80.56 g COD/L, accounting for 87.37 % of the soluble chemical oxygen demand), while also increasing the content of NH4+-N (2658.15 mg/L). Macrotranscriptomic sequencing showed that Anaerosalibacter, Amphibacillus, Wansuia, Clostridiisalibacter, unclassified Tissierellia, Massilibacterium, unclassified Bacteroidales, and Tissierellia were the key active microorganisms for VFA production. The expression abundance of functional enzymes and genes related to VFA production pathways increased during the fermentation. This study significantly advanced the practical application of VFA production from FW, offering both theoretical insights and bacterial resources.

Volatile fatty acids production from kitchen waste slurry using anaerobic membrane bioreactor via alkaline fermentation with high salinity: Evaluation on process performance and microbial succession

In this study, a pilot-scale anaerobic membrane bioreactor (AnMBR) was developed to continuously produce volatile fatty acids (VFAs) from kitchen waste slurry under an alkaline condition. The alkaline fermentation effectively suppressed methanogenesis, thus achieving high VFAs production of 60.3 g/L. Acetic acid, propionic acid, and butyric acid accounted for over 95.0 % of the total VFAs. The VFAs yield, productivity, and chemical oxygen demand (COD) recovery efficiency reached 0.5 g/g-CODinfluent, 6.0 kg/m3/d, and 62.8 %, respectively. Moreover, the CODVFAs/CODeffluent ratio exceeded 96.0 %, and the CODVFAs/NH3-N ratio through ammonia distillation reached up to 192.5. The microbial community was reshaped during the alkaline fermentation with increasing salinity. The membrane fouling of the AnMBR was alleviated by chemical cleaning and sludge discharge, and membrane modules displayed a sustained filtration performance. In conclusion, this study demonstrated that high-quality VFAs could be efficiently produced from kitchen waste slurry using an AnMBR process via alkaline fermentation.

Microbial composition play the leading role in volatile fatty acid production in the fermentation of different scale of corn stover with rumen fluid.

Rumen fluid is a natural and green biocatalyst that can efficiently degrade biomass into volatile fatty acid (VFA) used to produce value-added materials. But the essence of high degradation efficiency in the rumen has not been fully analyzed. This study investigated the contribution of substrate structure and microbial composition to volatile fatty acid production in the fermentation of corn stover. The ball milled corn stover were innovatively applied to ferment with the rumen fluid collected at different digestion times. Exogeneous cellulase was also added to the ruminal fermentation to further reveal the inner mechanism. With prolonged digestion time, the microbial community relative abundance levels of Bacteroidetes and Firmicutes increased from 29.98% to 72.74% and decreased from 51.76% to 22.11%, respectively. The highest VFA production of the corn stover was achieved via treatment with the rumen fluid collected at 24h which was up to 9508mg/L. The ball milled corn stover achieved high VFA production because of the more accessible substrate structure. The application of exogenous cellulase has no significant influence to the ruminal fermentation. The microbial community abundance contributed more to the VFA production compared with the substrate structures.

Marine Macroalgae in Rabbit Nutrition: In Vitro Digestibility, Caecal Fermentability, and Microbial Inhibitory Activity of Seven Macroalgae Species from Galicia (NW Spain)

The limitation on the prophylactic use of antibiotics in animal feed in Europe has critically challenged the rabbit meat industry, which urgently needs to find solutions. A feasible alternative could be using macroalgae in the diet to improve the gut health. This research studied seven species of marine macroalgae in four formats (dehydrated, enzymatically hydrolyzed, aqueous extract, and aqueous extract of hydrolyzed macroalgae) in order to select the most promising ones for their use in rabbit feed. Chemical composition, in vitro digestibility, in vitro caecal gas, total volatile fatty acid (VFA) production, and minimal inhibitory concentrations (MIC) against common pathogens were studied. All S. latissima products showed high caecal fermentability and VFA production, especially in both types of extracts. The H. elongata aqueous extract was remarkable due to its high in vitro butyrate production, which can be of great interest for improving gut health. The MIC results did not indicate any clear inhibition of the pathogens tested. The macroalgae tested appear to have a potentially prebiotic effect, rather than a direct antimicrobial activity. However, these results must be confirmed in vivo, in order to observe the real benefits of feeding macroalgae during the rabbit weaning period.

Self-fermented agro-wastes as antioxidant enriched maize grain replacer for sustainable animal feeding

Piles of agro-wastes resulting from quantum agro-industrial growth can be best utilized as animal feed, reducing envionmental impact by 50–95% depending on their nutritive value. However, palatability issues have limited their use, which can be addressed by combining them based on their nutritional value. Thus present three-phase study aimed to utilize these waste materials as animal feed by utilizing a self-fermentation approach. Six combinations of apple pomace (AP), spent mushroom compost (SMC) and wheat straw were self-fermented and analysed for quality in phase-I. Phase-II assessed the best combination obtained from phase-I as an antioxidant-enriched maize replacer (AEMR) by replacing maize (0–32%) in total mixed ration (TMR) under in-vitro ruminal conditions and two best TMR were assessed for nutrient utilization under metabolic trial. In phase-III, the three-month feeding effect of AEMR on the overall growth performance of male Gaddi goat kids was assessed. Findings showed that 85:7.5:7.5 proportion had optimal pH (3.92), significantly higher lactic acid (5.29 g 100−1 g DM), water soluble carbohydrates (2.29 g 100−1 g DM) and oxidative and microbial stability (Lactobacillus: 8.38 log10 cfu g−1). Fermentation also resulted in significant increase in antioxidant activities. AEMR exhibited a significant reduction in CH4 and NH3–N and higher volatile fatty acid production under in-vitro conditions at 32% level with no adverse effects on nutrient utilization. AEMR feeding also exhibited significantly improved feed conversion ratio (FCR) and serum lipid and oxidative profiles. In conclusion, AP and SMC in combined form can be utilized as an AEMR through self-fermentation at 32% level in TMR which may serve as a promising and sustainable approach for reducing environmental footprints and boosting eco-friendly livestock production.

Effect of mixing intensity on volatile fatty acids production in sludge alkaline fermentation: Insights from dissolved organic matter characteristics and functional microorganisms

Alkaline fermentation for volatile fatty acids (VFAs) production has shown potential as a viable approach to treat sewage sludge. The hydrolysis and acidogenesis of sludge are greatly influenced by mixing. However, the effects of mixing intensity on VFAs production in sludge alkaline fermentation (SAF) remain poorly understood. This study investigated the impacts of mixing intensity (30, 90 and 150 rpm continuous mixing, and 150 rpm intermittent mixing) on VFAs production, dissolved organic matter (DOM) characteristics, phospholipid fatty acid profiles and microbial population distribution in SAF. Results showed that 150 rpm continuous and intermittent mixing enhanced the hydrolysis of sludge, while 150 rpm intermittent mixing resulted in the highest VFAs production (3886 ± 266.1 mg COD/L). Analysis of fluorescent and molecular characteristics of DOM revealed that 150 rpm intermittent mixing facilitated the conversion of released DOM, especially proteins-like substances, into VFAs. The abundance of unsaturated and branched fatty acids of microbes increased under 150 rpm intermittent mixing, which could aid in DOM degradation and VFAs production. Firmicutes and Tissierella were enriched at 150 rpm intermittent mixing, which favored the maximum VFAs yield. Moreover, Firmicutes were found to be the key functional microorganisms influencing the yield of VFAs during SAF. This study provides an understanding about the mixing intensity effects on VFAs production during SAF, which could be helpful to improve the yield of VFAs.

Volatile fatty acid production in anaerobic fermentation of food waste saccharified residue: Effect of substrate concentration

In this study, food waste saccharified residue was used to produce volatile fatty acids (VFAs), and the effects of substrate concentration on VFA production, VFA composition, acidogenic efficiency, microbial community, and carbon transfer were investigated. Interestingly, chain elongation from acetate to n-butyrate played an important role with a substrate concentration of 200 g/L in the acidogenesis process. Results showed that 200 g/L was a suitable substrate concentration for both VFA and n-butyrate production, the highest VFA production, and n-butyrate composition were 280.87 mg COD/g vS and more than 90.00 %, respectively, and VFA/SCOD reached 82.39 %. Microbial analysis showed that Clostridium_Sensu_Stricto_12 promoted n-butyrate production by chain elongation. Carbon transfer analysis indicated that chain elongation made a contribution of 43.93 % to n-butyrate production. Totally 38.47 % of organic matter in food waste saccharified residue was further utilized. This study provides a new way for n-butyrate production with waste recycling and low cost.

Methane production by anaerobic co‐digestion of dairy manure and cassava wastes for energy recovery

AbstractBACKGROUNDIn this study, the anaerobic co‐digestion of cassava wastewater (CW), cassava bagasse (CB), and dairy manure (DM) was investigated to produce biogas with energy recovery. Mixture experimental design was performed to establish the proportion of each substrate, on the basis of total weight (%wt). The digestion was carried out under mesophilic conditions (35 °C) using a batch test.RESULTSThe best methane yields were 150.33 NmLCH4 gVS−1 (114.46 NmLCH4 gCOD−1) for the condition with 100% CW (C/N ratio = 14.04) and 130.43 normal mililiters of methane per gram of volatile solid (NmLCH4 gVS−1) and 93.84 normal mililiters of methane per gram of chemical oxygen demand (NmLCH4 gCOD−1) obtained with 66.7% DM, 16.7% CB and 16.7% CW (C/N ratio = 34.84). Experimental conditions containing high proportions of CB (above 33%) yielded high C/N ratios (above 52), resulting in low pH and high volatile fatty acid production, mainly acetic and butyric acids. Kinetics of methane production under optimal conditions were best described by Groot's multi‐stage model.CONCLUSIONAn energy balance for the best conditions were performed, leading to 452.86 MJ tonsubstrate−1 for a high DM content mixture experiment (DM = 66.7%, CB = 16.7%, CW = 16.7%); thereby showing that anaerobic co‐digestion can replace 22.5% of the energy demanded by cassava processing industry. © 2022 Society of Chemical Industry (SCI).

Combined modifications of CaO and liquid fraction of digestate for augmenting volatile fatty acids production from rice straw: Microbial and proteomics insights

The modification sequence of chemical (CaO) and biological (liquid fraction of digestate, LFD) for augmenting volatile fatty acids (VFAs) production from rice straw was investigated in this study. The coupling order of the modifiers influenced acidification performance, and simultaneous modification (CaO-LFD) was superior to other modes. The highest VFAs production was obtained in CaO-LFD, 51% higher than that in the LFD-first additional modification. The CaO-LFD demonstrated the highest selectivity of acetate production, accounting for 79% of the total VFAs. In addition, CaO-LFD modification changed the direction of the domestication of fermentative bacteria and increased populations of the key anaerobes (Atopostipes sp.) responsible for acidification. The synergistic effect of CaO and LFD was revealed, namely, the effective function of CaO in degrading recalcitrant rice straw, the promotion of transport/metabolism of carbohydrates and acetogenesis by LFD.

Optimization of volatile fatty acid production by sugarcane vinasse dark fermentation using a response surface methodology. Links between performance and microbial community composition

The recovery of volatile fatty acids (VFA) from waste and wastewaters has recently been pointed out as a promising strategy for adding value to residues, which characterizes the carboxylate platform. The exploitation of sugarcane vinasse within the carboxylate platform is still an incipient approach and has to be further investigated to determine the conditions which optimize the process. This study aimed to optimize the production of VFA (as the sum of concentrations of acetic, butyric and propionic acids) through the dark fermentation (DF) of sugarcane vinasse. A central composite design (CCD, factorial 22) and response surface methodology (RSM) regarding two independent variables, namely, temperature (33–47 °C) and initial pH (7.1–9.9), was performed. Experiments were conducted in batch reactors using diluted vinasse (COD = 10 gL−1) without nutrient supplementation. The results showed that the production of VFA reached a peak of 2,980 mgL−1 (as the sum of VFA produced), yield of 332 mg-CODVFAg−1CODtinicial and productivity of 619 mg-CODL−1d−1) at 39.6 °C and initial pH of 8.8. The microbial community analysis performed by 16S rRNA gene amplicon sequencing showed that organisms from the families Clostridiaceae, Enterobacteriaceae and Lachnospiraceae, and Veillonellales-Selenomonadales order prevailed during higher VFA production, whilst the presence of Desulfotomaculales and Lactobacillaceae families could be associated with VFA consumption and lactic acid production, respectively. These results provide a basis for vinasse management within the carboxylate platform context, potentially diversifying the product portfolio of sugarcane biorefineries.

Improvement of Rumen Fermentation Efficiency Using Different Energy Sources: In Vitro Comparison between Buffalo and Cow

During haymaking and ensilage, a significant loss of sugars occurs. The addition of a total mixed ratio (TMR) with a liquid feed might provide promptly utilisable energy and recover the nutrients lost during the conservation. Interesting results were already obtained by including liquid feed in a TMR in a dairy cow. However, the possibility to also utilize them in Italian Mediterranean buffalo is not yet supported by data. This study aimed to evaluate the in vitro fermentation characteristics and kinetics of different types of liquid feed, utilising bovine and buffalo rumen liquor as inocula. TMR supplemented with 0.025 g of four different liquid feeds was incubated with the TMR as control with buffalo and bovine rumen fluid using in vitro gas production technique. Considering bovine inoculum, all the experimental diets showed lower organic matter degradability and higher volatile fatty acid production than control TMR, while with buffalo rumen liquor, significant differences were observed between experimental and control diets in terms of gas production and fermentation kinetics. The tested liquid feeds can have different fermentation patterns depending on their ingredients and compositions. Supplementing liquid feeds to a standard diet seems to provide a source of energy that improves fermentation. No negative effects were observed on the in vitro fermentation at the dosage utilised.

Dark Fermentation of Sweet Sorghum Stalks, Cheese Whey and Cow Manure Mixture: Effect of pH, Pretreatment and Organic Load

The aim of this study was to determine the optimal conditions for dark fermentation using agro-industrial liquid wastewaters mixed with sweet sorghum stalks (i.e., 55% sorghum, 40% cheese whey, and 5% liquid cow manure). Batch experiments were performed to investigate the effect of controlled pH (5.0, 5.5, 6.0, 6.5) on the production of bio-hydrogen and volatile fatty acids. According to the obtained results, the maximum hydrogen yield of 0.52 mol H2/mol eq. glucose was measured at pH 5.5 accompanied by the highest volatile fatty acids production, whereas similar hydrogen productivity was also observed at pH 6.0 and 6.5. The use of heat-treated anaerobic sludge as inoculum had a positive impact on bio-hydrogen production, exhibiting an increased yield of 1.09 mol H2/mol eq. glucose. On the other hand, the pretreated (ensiled) sorghum, instead of a fresh one, led to a lower hydrogen production, while the organic load decrease did not affect the process performance. In all experiments, the main fermentation end-products were volatile fatty acids (i.e., acetic, propionic, butyric), ethanol and lactic acid.

Continuous Volatile Fatty Acid Production From Acid Brewery Spent Grain Leachate in Expanded Granular Sludge Bed Reactors

The production of volatile fatty acids (VFAs) in expanded granular sludge bed (EGSB) reactors using leachate from thermal diluted acid hydrolysis of brewery spent grain was evaluated. Partial inhibition of the anaerobic digestion process to induce VFA accumulation was achieved by applying a high organic loading rate [from 15.3 to 46.0 gCOD/(L·day)], and using a feed with an inlet concentration of 15 g/L total carbohydrates. Two EGSB reactors were operated under identical conditions, both inoculated with the same granular sludge. However, granular sludge in one reactor (R1) was subsequently disaggregated to flocculent sludge by a pH shock, whereas granules remained intact in the other reactor (R2). The hydraulic retention time (HRT) of both reactors was decreased from 36 to 24, 18 and 12 h. The main fermented compounds were acetic acid, butyric acid, propionic acid and ethanol. Despite fluctuations between these products, their total concentration was quite stable throughout the trial at about 134.2 (±27.8) and 141.1 (±21.7) mmol/L, respectively, for R1 and R2. Methane was detected at the beginning of the trial, and following some periods of instability in the granular sludge reactor (R2). The hydrogen yield increased as the HRT decreased. The highest VFA production was achieved in the granular sludge reactor at a 24 h HRT, corresponding to 120.4 (±15.0) mmol/L of VFAs. This corresponded to an acidification level of 83.4 (±5.9) g COD of VFA per 100 gram of soluble COD.

Bioconversion of food waste to volatile fatty acids: Impact of microbial community, pH and retention time

Bio-based production of materials from waste streams is a pivotal aspect in a circular economy. This study aimed to investigate the influence of inoculum (three different sludge taken from anaerobic digestors), pH (5 & 10) and retention time on production of total volatile fatty acids (VFAs), VFA composition as well as the microbial community during anaerobic digestion of food waste. The highest VFA production was ∼22000 ± 1036 mg COD/L and 12927 ± 1029 mg COD/L on day 15 using the inoculum acclimated to food waste at pH 10 and pH 5, respectively. Acetic acid was the dominant VFA in the batch reactors with initial alkaline conditions, whereas both propionic and acetic acids were the dominant products in the acidic condition. Firmicutes, Chloroflexi and Bacteroidetes had the highest relative abundance in the reactors. VFA generation was positively correlated to the relative abundance of Firmicutes.

Bioconversion of potato waste by rumen fluid from slaughterhouses to produce a potential feed additive rich in volatile fatty acids for farm animals

Potato waste, usually derived from storage and processing of potatoes, is an agricultural byproduct already used as a feed for ruminants. Its fermentation using rumen fluid, another waste product obtainable from slaughterhouses, is expected to valorize both products. Bioconversion of potato waste using rumen fluid was followed by measuring volatile fatty acids (VFAs), pH, and the extent of digestion of the potato waste as well as the nutrient composition of the solid residues after fermentation. Different ratios (1:2, 2:2, 3:2 and 4:2 w dry matter/v) of potato waste to rumen fluid were fermented in a DaisyII incubator for 12 or 24 h at 39 °C. Depending on the fermentation time and the dry mass of potato waste used for fermentation, a digestion rate between 31 and 78.8% of the potato waste (solid phase of the fermentation mixture) was obtained. Fermentation resulted also in an up to 2.5-fold increase in the concentration of VFAs in the liquid phase of the fermentation mixture. During fermentation, the pH value of the fermentation mixture dropped from 6.95 to about 4.0 (P < 0.05). The highest digestion rate and the highest VFA production were obtained with 100 g potato waste and a 24 h fermentation time. Furthermore, the residual solid residues after fermentation had higher protein and fat contents compared to the potato waste used for fermentation (P < 0.05). Based on the results obtained, potential applications of both potato wastes and rumen fluids have been identified. The used method for bioconversion is suggested for scale-up purposes.

Novel strategy to stimulate the food wastes anaerobic fermentation performance by eggshell wastes conditioning and the underlying mechanisms

A novel strategy of utilizing eggshell wastes to condition food wastes (FW) anaerobic fermentation for efficient volatile fatty acids (VFAs) production was proposed. The VFAs production, especially acetic and butyric acids, was significantly accelerated and enhanced by eggshell conditioning. The enhanced effects were dose-dependent and improved by approximate 13-folds with optimal dose when compared with that in control. Eggshell conditioning synchronously enhanced the FW solubilization, hydrolysis and acidification steps, resulting in high VFAs production. Further analysis found that the environmental conditions (i.e., pH, ORP, trace elements, etc.) were significantly improved by eggshell conditioning. Hence, the microbial activities (i.e., hydrolases, acidogenic enzymes and ATP) and functional fermentative bacteria related with VFAs generation were enhanced and enriched. The massive accumulation of H2 and CO2 in eggshell conditioning reactors stimulated the homoacetogenesis, which also contributed to the VFAs promotion. Metatranscriptomic analysis indicated that 3 carbohydrate-active enzymes (Glycoside hydrolases, carbohydrate esterases and carbohydrate binding modules), which participate in carbohydrates and energy metabolisms, were improved. Moreover, 10 kinds of synthetase, hydrolase and oxidoreductase genes, and 4 kinds of acetyl-CoA carboxylase genes that distributed throughout the fatty acid biosynthesis pathway (especially the acetic and butyric acids generation) were all enhanced. It was the highly expressed genes and metabolic enzymes that contributed to high microbial activities and subsequent efficient VFAs production in eggshell conditioning reactors. Besides, the improvement of dewaterability and phosphorus fixation in fermentation residues further increased the added-value of conditioning FW fermentation by eggshell wastes.

A comprehensive study of volatile fatty acids production from batch reactor to anaerobic sequencing batch reactor by using cheese processing wastewater

Volatile fatty acids (VFAs) has great potential for closed-loop production in dairy industries via resource recovery from waste-streams. In the current study, the transition of VFA production from batch reactor to anaerobic sequencing batch reactor (ASBR) by using cheese industry wastewater under alkali pH was evaluated with respect to seed sludge structure, microbial diversity and reactor type. The transition from the batch reactor to the ASBR demonstrated that the maximum VFA production yield (g COD/g SCOD) was comparable in two reactors (batch: 0.97; ASBR: 0.94), whereas, the dominant acid type was different (batch: 49% lactic acid; ASBR: 80% propionic acid). There was a significant correlation between the productions of butyric acid with Gracilibacteraceae and Desulfovibrionaceae; propionic acid with Desulfovibrionaceae and Synergistaceae; lactic acid with Pseudomonadaceae and Rhodocyclaceae. The high VFA production efficiency can be achieved by long term reactor operation, which enables the shift from industrial waste-streams to biorefineries.

Achieving high volatile fatty acid production from raw henna (Lawsonia inermis) biomass at mild alkaline conditions

Strong alkaline hydrolysis is usually employed to enhance fermentative production of volatile fatty acid (VFA) from plant biomass. However, this method is not ecofriendly due to the large consumption of energy/chemicals. In this study, a special henna plant biomass (Lawsonia inermis) containing the chemical compound lawsone, served as a fermentation substrate (without any pretreatment) for high VFA production under mild alkaline conditions (pH 9). The results indicated that the fermentative VFA production was 0.31 g per gram of henna biomass, which was comparable to other plant biomass pretreated with hydrothermal, strong acidic, and/or alkaline methods. Acetate was the main VFA component, and accounted for 69% to 82% of the COD fraction. The soluble lawsone concentration in the fermentation system ranged from 8.5 mg/L to 31.2 mg/L, which mediated the potential fermentation process. Thus, in this study henna was established as a feasible plant biomass for high fermentative VFA production in mild alkaline conditions.

Achieving high volatile fatty acid production from raw henna (Lawsonia inermis) biomass at mild alkaline conditions

Strong alkaline hydrolysis is usually employed to enhance fermentative production of volatile fatty acid (VFA) from plant biomass. However, this method is not ecofriendly due to the large consumption of energy/chemicals. In this study, a special henna plant biomass (Lawsonia inermis) containing the chemical compound lawsone, served as a fermentation substrate (without any pretreatment) for high VFA production under mild alkaline conditions (pH 9). The results indicated that the fermentative VFA production was 0.31 g per gram of henna biomass, which was comparable to other plant biomass pretreated with hydrothermal, strong acidic, and/or alkaline methods. Acetate was the main VFA component, and accounted for 69% to 82% of the COD fraction. The soluble lawsone concentration in the fermentation system ranged from 8.5 mg/L to 31.2 mg/L, which mediated the potential fermentation process. Thus, in this study henna was established as a feasible plant biomass for high fermentative VFA production in mild alkaline conditions.

Effect of different heat treatments of inoculum on the production of hydrogen and volatile fatty acids by dark fermentation of sugarcane vinasse

Vinasse the main residue of bioethanol production can be used as a substrate in microbiological processes to obtain value-added products. However, it is necessary to use a microbial consortium with high capacity to produce hydrogen (H2) and volatile fatty acids (VFA). There is no consensus on the best inoculum pretreatment to eliminate hydrogen-consuming bacteria. Thus, the present study evaluated the influence of three methods of heat pretreatment of the inoculum: T1 = 90 °C/10 min; T2 = 105 °C/120 min; T3 = 121 °C/20 min for the production of H2 and VFA using vinasse as substrate. The effect of the concentration of vinasse and the initial pH (5, 6 and 7) were also evaluated. The highest hydrogen production (821.34 mL) and yield (4.75 mmol H2 g−1 COD) was obtained using undiluted vinasse at pH 6 and T1 pretreatment. The highest number of copies of the Fe-hydrogenase genes confirms the higher H2 production. The presence of Clostridium and facultative anaerobic microorganisms Bacillus and Enterobacter in the microbial consortia were confirmed by isolation and PCR-DGGE. The highest production of VFA was obtained at pH 7 and T3 pretreatment. This study showed that dark fermentation could be driven by the inoculum pretreatment and pH selecting different process either for the production of H2 or VFA from vinasse in natura, without addition of supplements.