Dark fermentation of starch factory wastewater with acid- and base-treated mixed microorganisms for biohydrogen production

Biohydrogen (Bio-H₂) can be produced from starch factory wastewater and mixed microorganisms using dark fermentation. Acidic and basic chemicals were used to treat the microorganisms to select the hydrogen (H₂)-producing culture. The experiment used a 120 mL bioreactor at 35 °C and the operation commenced with the initial pH level of wastewater in the pH range 4–7 in batch mode. The bacteria:chemical oxygen demand (COD) ratio was 0.2. The initial pH level of the wastewater in the fermentation process affected the H₂ yield and the specific hydrogen production rate (SHPR). For acid-treated bacteria, the maximum H₂ yield and SHPR were produced at an initial pH of 6.5. The maximum H₂ yield and SHPR were 138 mL/g COD degraded and 7.42 mL/g cells?h, respectively. For the base-treated bacteria, the maximum H₂ yield and SHPR were produced at initial pH of 6.5 and pH 7, respectively. The maximum H₂ yield and SHPR were 182 mL/g COD degraded and 25.60 mL/g cells?h, respectively. The COD degradation efficiency levels were 16 and 20% for acid- and base-treated bacteria, respectively. The digested wastewater remained acidic at pH 4.79–4.83. Throughout the study, no methane gas was observed in the gas mixture produced.     

A spectrophotometric method for quantitative determination of xylose in fermentation medium

Monitoring the consumption of sugars during fermentation is a key to optimizing product formation and maintaining a healthy environment for microorganisms. Difficulty arises in the availability of a rapid, inexpensive and sensitive method for the detection of sugars because fermentation media are a complex mix of nutrients, cell debris, waste and target products. A method involving reaction-based UV-Vis spectrophotometry for the quantitative determination of xylose as the target sugar was developed. Factors affecting xylose concentration measurements such as hydrochloric acid concentration, heating time and the amount of Fe³⁺ catalyst were investigated. A continuous scan revealed the working wavelength to be 671 nm. The effect of other components in the fermentation broth was found to be negligible. Absorbance shows a linear relationship with xylose concentration within a range of 0.1-0.5 g/L. Xylose concentrations from fermentation samples obtained at specific time intervals (0-168 h) were determined with the method and compared with YSI 2700, an enzyme electrode, HPLC-ELSD method, currently a common technique for measuring xylose and GC aldononitrile sugar derivatization method. Dilution is necessary for comparable xylose concentrations with YSI 2700 and HPLC-ELSD. Xylose concentration measurements obtained with the UV-Vis spectrophotometric method although quantitatively comparable to HPLC-ELSD xylose measurements were easily and conveniently obtained compared to YSI 2700, HPLC-ELSD and GC derivatization methods.     

Statistical optimization of factors affecting biohydrogen production from xylose fermentation using inhibited mixed anaerobic cultures

The role of different chemical and physical factors in enhancing biohydrogen production from xylose using a mixed anaerobic culture was examined under mesophilic conditions. A fractional factorial design (FFD) 3^(k–p) was used to optimize pH, the oleic acid (OA) concentration and the biomass concentration. The FFD analysis indicated that the hydrogen (H₂) yield was affected by 3 single factors as well as by 2 factor interactions. Under optimum conditions (1600 mg L⁻¹ of oleic acid (OA) and 1900 mg L⁻¹ VSS and pH 6.7), the H₂ yield reached 2.64 ± 0.12 mol mol⁻¹ of xylose (80% of the theoretical yield). Based on the ANOVA and Pareto chart analysis, the linear and quadratic OA and pH terms were significant and the linear and quadratic VSS terms were insignificant. Normally distribution of the residuals was confirmed from the Anderson-Darling (AD) plot. The studentized residuals versus the predicted values plot clearly demonstrated that the data points were randomly scattered.     

The addition of hydrolyzed rice straw in xylose fermentation by Pichia stipitis to increase bioethanol production at the pilot-scale

The pretreatment of agricultural biomass by diluted acid is often employed to facilitate the release of monosaccharide for the subsequent enzyme hydrolysis for lignocellulosic ethanol production. However, furfural and hydroxymethylfurfural are usually generated and markedly decrease the yield of pentose fermentation during this pretreatment. In the present study, the enhancement of lignocellulosic ethanol production was successfully demonstrated at pilot scale with extra addition of hydrolyzed rice straw into pentose fermentation by Pichia stiptis. This way has resulted into the increase of P. stiptis cell mass was shown to play a positive role. The ethanol yield, 0.45 g_p/g_s, with the addition of hydrolyzed rice straw in hemicellulosic hydrolysate from plywood, bagasse and bamboo were increase 20–51% to demonstrate the applicability of this technology in a variety of lignocellulosic ethanol processes due to the efficient conversion of xylose.     

Sequential generation of hydrogen and methane from xylose by two-stage anaerobic fermentation

The sequential generation of hydrogen and methane from xylose by two-stage anaerobic fermentation was investigated for the first time in this study. The effects of substrate concentration, bacteria domestication and nitrogen source on hydrogen yield were studied in the first stage. The genetic characterization of the 16S rDNA was used to analyze the flora of strains domesticated with xylose and glucose. The maximum hydrogen yield is 190.6 ml H₂/g xylose when the xylose feedstock concentration is 1% (w/v), hydrogenogens are domesticated with xylose and yeast extract is used as nitrogen source. The soluble metabolite byproducts (SMB) from the hydrogen-producing stage were reutilized by methanogens to produce methane in the second stage. Over 98 wt % of acetate and butyrate in the SMB are reutilized to give a methane yield of 216.5 ml CH₄/g xylose. The sequential generation of hydrogen and methane from xylose markedly increases the energy conversion efficiency to 67.5%.     

Optimization of very high gravity fermentation process for ethanol production from industrial sugar beet syrup

In order to reduce production costs and environmental impact of bioethanol from sugar beet low purity syrup 2, an intensification of the industrial alcoholic fermentation carried out by Saccharomyces cerevisiae is necessary. Two fermentation processes were tested: multi-stage batch and fed-batch fermentations with different operating conditions. It was established that the fed-batch process was the most efficient to reach the highest ethanol concentration. This process allowed to minimize both growth and ethanol production inhibitions by high sugar concentrations or ethanol. Thus, a good management of the operating conditions (initial volume and feeding rate) could produce 15.2% (v/v) ethanol in 53 h without residual sucrose and with an ethanol productivity of 2.3 g L h⁻¹.     

Anthrahydroquinone-2,6-disulfonate increases the rate of hydrogen production during Clostridium beijerinckii fermentation with glucose,xylose, and cellobiose

Fermentative hydrogen production is considered a reasonable alternative for generating H₂ as an energy carrier for electricity production using hydrogen fuel cells. The kinetics of hydrogen production from glucose, xylose and cellobiose were investigated using pure culture Clostridium beijerinckii NCIMB 8052. Adding anthrahydroquinone-2,6-disulfonate (AH₂QDS) at concentrations ranging from 100 μM to 500 μM increased the hydrogen production rates from 0.80 to 1.35 mmol/L-hr to 1.20–2.70 mmol/L-hr with glucose, xylose, or cellobiose as the primary substrates. AH₂QDS amendment also increased the substrate utilization rate and biomass growth rate by at least two times. These findings suggest that adding hydroquinone reducing equivalents influence cellular metabolism with hydrogen production rate, substrate utilization rate, and growth rate being simultaneously affected. Resting cell suspensions were conducted to investigate the influence of AH₂QDS on the hydrogen production rate from glyceraldehyde 3-phosphate, which is a shared intermediate in both glycolysis and pentose phosphate pathway. Data demonstrated that hydrogen production rate increased by 1.5 times when glyceraldehyde 3-phosphate was the sole carbon source, suggesting that the hydroquinone may alter reactions starting with or after glyceraldehyde 3-phosphate in central metabolism. These data demonstrate that adding hydroquinones increased overall metabolic activity of C. beijerinckii. This will eventually increase the efficiency of industrial scale production once appropriate hydroquinone equivalents are identified that work well in large-scale operations, since fermentation rate is one of the two critical factors (production rate and yield) influencing efficiency and cost.     

Hydrogen production from acidogenic food waste fermentation using untreated inoculum: Effect of substrate concentrations

The effect of substrate concentrations (0, 7.5, 15, 22.5, 30, and 37.5 g-VS/L) on hydrogen production from heat-treated and fresh food waste (FW) using untreated inoculums was investigated in this work. The highest hydrogen yield (75.3 mL/g-VS) was obtained with heat-treated FW at 15 g-VS/L. Lower substrate content could not provide enough organic matter for hydrogen fermentation, while higher substrate concentrations shifted the metabolic pathways from hydrogen fermentation to lactic acid fermentation by enriching the lactic acid bacteria (LAB), which lowered the slurry pH and decreased enzyme activity, resulting in a lower chemical oxygen demand (COD), volatile solid (VS), carbohydrate removal rate, and hydrogen yield. Compared with fresh FW, heat-treated FW is preferred for biohydrogen process with acetate as the main organic product. Additionally, at the optimal concentration (15 g-VS/L) using fresh FW, lactic acid is first accumulated and then degraded to produce hydrogen with butyrate as the main metabolite.     

Reactor start-up strategy as key for high and stable hydrogen production from cheese whey thermophilic dark fermentation

Using the right start-up strategy can be vital for successful hydrogen production from thermophilic dark fermentation (55 °C), but it needs to be affordable. Hence, three start-up strategies modifying only influent concentration and temperature were assessed in a reactor fed with cheese whey: (i) high temperature (55 °C) and a high organic loading rate (OLR_A - 15 kgCOD m^?3 d^?1) right at the beginning of the operation; (ii) slowly increasing temperature up to 55 °C using a high OLR_A and (iii) slowly increasing temperature and OLR_A up to the desired condition. Strategy (iii) increased hydrogen productivity in 39% compared to the others. The combination of high temperature and low pH thermodynamically favored H₂ producing routes. Synergy between Thermoanaerobacterium and Clostridium might have boosted hydrogen production. Three reactors of 41 m³ each would be needed to treat 3.4 × 10³ m³ year^?1 of whey (small-size dairy industry) and the energy produced could reach 14 MWh month^?1.     

Effect of food to microorganisms (F/M) ratio on biohythane production via single-stage dark fermentation

Hythane is a mixture of hydrogen and methane gases which are generally produced in separate ways. This work studied mesophilic biohythane gas (H₂+CH₄+CO₂) production in a bioreactor via single-stage dark fermentation. The fermentation was conducted in batch mode using mixed anaerobic microflora and food waste and condensed molasses fermentation soluble to elucidate the effects of food to microorganisms (F/M) ratio (ranging from 0.2 to 38.2) on gas production, metabolite variation, kinetics and biohythane-composition indicator performances. The experimental results indicate that the F/M ratio and fermentation time affect biohythane production efficiency with values of peak maximum hydrogen production rate 9.60 L/L-d, maximum methane production rate 0.72 L/L-d, and hydrogen yield (HY) of 6.17 mol H₂/kg COD_added. Depending on the F/M ratios, the H₂, CH₄ and CO₂ biogas components were 10–60%, 5–20% and 35–70%, respectively. Prospects for the further real application for single-stage biohythane fermentation based on the experimental data are proposed. This work characterizes an important reactor operation factor F/M ratio for innovative single-stage dark fermentation.     

Light fermentation of dark fermentation effluent for bio-hydrogen production by different Rhodobacter species at different initial volatile fatty acid (VFA) concentrations

Three different Rhodobacter sphaeroides (RS) strains (RS–NRRL, RS–DSMZ and RS–RV) and their combinations were used for light fermentation of dark fermentation effluent of ground wheat containing volatile fatty acids (VFA). In terms of cumulative hydrogen formation, RS–NRRL performed better than the other two strains producing 48 ml H₂ in 180 h. However, RS–RV resulted in the highest hydrogen yield of 250 ml H₂ g⁻¹ TVFA. Specific hydrogen production rate (SHPR) with the RS–NRRL was also better in comparison to the others (13.8 ml H₂ g⁻¹ biomass h⁻¹). When combinations of those three strains were used, RS–RV + RS–DSMZ resulted in the highest cumulative hydrogen formation (90 ml H₂ in 330 h). However, hydrogen yield (693 ml H₂ g⁻¹ TVFA) and SHPR (12.1 ml H₂ g⁻¹ biomass h⁻¹) were higher with the combination of the three different strains. On the basis of Gompertz equation coefficients mixed culture of the three different strains gave the highest cumulative hydrogen and formation rate probably due to synergistic interaction among the strains. The effects of initial TVFA and NH₄–N concentrations on hydrogen formation were investigated for the mixed culture of the three strains. The optimum TVFA and NH₄–N concentrations maximizing the hydrogen formation were determined as 2350 and 47 mg L⁻¹, respectively.     

Fungal solid-state fermentation of food waste for biohydrogen production by dark fermentation

The dark fermentation process was evaluated for biohydrogen production from food waste through fungal solid-state fermentation (SSF). Three fungal cultures (one strain of Aspergillus tubingensis and two strains of Meyerozyma caribbica) were compared, being A. tubingensis the best hydrolyser culture for releasing soluble carbohydrates. The biochemical hydrogen potential of food waste hydrolysate (FWH) at different substrate-inoculum ratios obtained a lower hydrogen yield than untreated food waste (RFW). The highest hydrogen yield value corresponded to treatments RFW-20 and RFW-30 with 77.0 ± 2.6 and 76.9 ± 1.4 mL H₂ normalized by per gram volatile solid added (NmL H₂/gVS_added), respectively. The microbial community of food waste was analysed, being detected lactic-acid bacteria genera as Latilactobacillus and Leuconostoc. The presence of actively growing bacteria during the SSF could explain the lowest hydrogen yield (20.1–36.0 NmL H₂/gVS_added) in the FWH treatment due to the substrate competition between lactic-acid bacteria and hydrogen-producing bacteria, where the lactic-acid bacteria were favoured by their faster growth rate.     

Sulfate-reducing bacteria as new microorganisms for biological hydrogen production

Sulfate-reducing bacteria (SRB) have an extremely high hydrogenase activity and in natural habitats where sulfate is limited, produce hydrogen fermentatively. However, the production of hydrogen by these microorganisms has been poorly explored. In this study we investigated the potential of SRB for H₂ production using the model organism Desulfovibrio vulgaris Hildenborough. Among the three substrates tested (lactate, formate and ethanol), the highest H₂ production was observed from formate, with 320 mL L⁻¹_medium of H₂ being produced, while 21 and 5 mL L⁻¹_medium were produced from lactate and ethanol, respectively. By optimizing reaction conditions such as initial pH, metal cofactors, substrate concentration and cell load, a production of 560 mL L⁻¹_medium of H₂ was obtained in an anaerobic stirred tank reactor (ASTR). In addition, a high specific hydrogen production rate (4.2 L g⁻¹_dcw d⁻¹; 7 mmol g⁻¹_dcw h⁻¹) and 100% efficiency of substrate conversion were achieved. These results demonstrate for the first time the potential of sulfate reducing bacteria for H₂ production from formate.     

Fed-batch Saccharomyces cerevisiae fermentation of hydrolysate sugars: A dynamic model-based approach for high yield ethanol production

The efficient fermentation of hydrolyzed sugars from lignocellulosic biomass feedstock to ethanol remains a complex multi-parametric problem. Thus, in the present study, an advanced structured dynamic model for the simulation of the fermentative ethanol production from hydrolysate sugars is developed. The model is combined with a statistical experimental design to determine an optimal operating strategy that maximizes ethanol production and serves for the systematic evaluation of critical process variables. In particular, the effects of various operating conditions and feeding strategies on the dynamic behavior of batch and fed-batch fermentation processes are explored. The deviation from the desired product or the metabolic inhibition of ethanol production are related with the applied environmental conditions and substrate and product inhibition phenomena. The operating strategy, designed with the assistance of the mathematical tools proposed in this study, includes an exponential addition policy of substrate. This strategy is experimentally proved to enhance the final product concentration, raising the ethanol productivity to 2.27 g L⁻¹ h⁻¹ and the ethanol yield to 53.5% of the maximum theoretical value. Moreover, the simulated strategies were in excellent agreement with the experimental results obtained from the real process using low and high glucose initial concentration, under batch and fed-batch conditions, in both flask- and bioreactor-scale cultivations, proving the model's predictive and optimization capabilities. Further improvement of process performance is expected when combining the proposed dynamic model with advanced optimization algorithms to derive the optimal bioprocess operating strategy.     

Enhanced hydrogen production from xylose and bamboo stalk hydrolysate by overexpression of xylulokinase and xylose isomerase in Klebsiella oxytoca HP1

Biofuels production from lignocellulose hydrolysates by microbe fermentation has merited attention because of the mild reaction conditions involved and the clean nature of the process. In this work, xylulokinase (XK) and xylose isomerase (XI) were overexpressed in Klebsiella oxytoca HP1 to enhance hydrogen production by the fermentation of xylose. The recombinant strains exhibited higher enzyme activity of XI or XK compared with the wild strain. Hydrogen production from pure xylose, xylose/glucose mixtures and bamboo stalk hydrolysate was significantly enhanced with the overexpression of XI and XK in K. oxytoca HP1 in terms of total hydrogen yield (THY), hydrogen yield per mole substrate (HYPM) and hydrogen production rate (HPR). The HYPM of K. oxytoca HP1/xylB and K. oxytoca HP1/xylA reached 1.93 ± 0.05 and 2.46 ± 0.05 mol H₂/mol xylose, respectively in pure xylose, while the value for the wild strain was 1.68 ± 0.04 mol H₂/mol xylose. The xylose consumption rate (XCR) for the recombinant strain was significantly higher than that for the wild strain, particularly in the early stage of fermentation. Relative to the wild type, hydrogen yield (HY) from 1 g of preprocessed bamboo powder of HP1/xylB and HP1/xylA increased by 33.04 and 41.31%, respectively. It was concluded that overexpression of XK or XI was able to promote hydrogen production from xylose and xylose/glucose mixtures by simultaneously increasing the utilization efficiency of xylose and weakening the inhibitory effect of glucose on xylose use. In addition, the results indicated that overexpression technology was an effective way to further increase hydrogen production from lignocellulosic hydrolysates.     

Challenges for biohydrogen production via direct lignocellulose fermentation

Direct cellulose fermentation by cellulolytic anaerobic bacteria offers potential to generate renewable hydrogen (H₂) from inexpensive “waste” cellulosic feedstocks. The rates and yields of H₂ production via direct cellulose fermentation are low and must be increased significantly if this technology is to become a viable method for generating usable H₂. A much more comprehensive understanding of the relationships between gene and gene product expression, end-product synthesis patterns, and the factors that regulate carbon and electron balance, within the context of the bioreactor conditions must be achieved if we are to improve molar yields of H₂ during cellulose fermentation. Strategies to increase yields of H₂ production from cellulose include manipulation of carbon and electron flow via end-product inhibition (metabolic shift), metabolic engineering at the genetic level, synergistic co-cultures, and bioprocess engineering and bioreactor designs that maintain a neutral pH during fermentation and ensure rapid removal of H₂ and CO₂ from the aqueous phase.     

Bio-hydrogen behavior of suspended and attached microorganisms in anaerobic fluidized bed

An anaerobic fluidized bed reactor (110 L AnFBR) which kitchen waste (KW) was fed as the major substrate and different weight fractions of napiergrass dregs as the supplemental ones for bio-hydrogenation at long hydraulic retention time of 7.3 days was established. Two types of microorganisms could be distinguished by their life surroundings in the microbial community of the AnFBR: one is suspended growth, the other is attached growth. In order to evaluate the biohydrogen potential and kinetic characteristics of the two microbial growths in the AnFBR, a series of biodegradation batch experiments were conducted as the biochemical hydrogen potential test. The suspended microbe had obvious bio-hydrogen production after 3.6 h of lag phase, and the maximum specific hydrogen production rate achieved at 2.65 mmol/g-VSS-hr; the attached microbe stayed longer lag phase about 20 h and the maximum specific hydrogen production rate reached up to 1.06 mmol/g-VSS-hr. The metabolism study was investigated with volatile fatty acids conversion with initial, middle and final constitutes. The huge amount of lactic acid degradation of suspended growth (about 6000 mg/L degradation) displayed a special phenomenon in fermentative hydrogen production. Scanning electron microscope (SEM) was used to observe the morphology of two cultures. We also performed a terminal restriction fragment length polymorphism (T-RFLP) analysis for the diverse microbial identification.     

Hydrogen production by microorganisms and its application in a PEMFC

This paper compares some important parameters obtained in the hydrogen production between the photosynthetic microorganisms Spirulina maxima 2342 and Scenedesmus obliquus 39. It is also reported the employment of hydrogen produced in this study (m³ s^?1) in a proton exchange membrane fuel cell (PEMFC) for electricity production. A comparison is also made between the electric current generated and the final dry biomass (mA mg^?1) under specific experimental conditions. In this study, the electric current generated in the PEMFC for a period of 200 min under the light intensity of 150 µE m^?2 s^?1 and agitation was monitored. With the average current generated by the fuel cell the hydrogen produced by each microorganism is determined. The chromatography method was used to confirm the presence of hydrogen produced by each microorganism which was fed to the PEMFC. Copyright © 2011 John Wiley & Sons, Ltd.     

Enhancement of hydrogen production rate by high biomass concentrations of Thermotoga neapolitana

The objective of this study was to enhance the hydrogen production rate of dark fermentation in batch operation. For the first time, the hyperthermophilic pure culture of Thermotoga neapolitana cf. Capnolactica was applied at elevated biomass concentrations. The increase of the initial biomass concentration from 0.46 to 1.74 g cell dry weight/L led to a general acceleration of the fermentation process, reducing the fermentation time of 5 g glucose/L down to 3 h with a lag phase of 0.4 h. The volumetric hydrogen production rate increased from 323 (±11) to 654 (±30) mL/L/h with a concomitant enhancement of the biomass growth and glucose consumption rate. The hydrogen yield of 2.45 (±0.09) mol H₂/mol glucose, the hydrogen concentration of 68% in the produced gas and the composition of the end products in the digestate, i.e. 62.3 (±2.5)% acetic acid, 23.5 (±2.9)% lactic acid and 2.3 (±0.1)% alanine, remained unaffected at increasing biomass concentrations.     

Inoculum pretreatment differentially affects the active microbial community performing mesophilic and thermophilic dark fermentation of xylose

The influence of different inoculum pretreatments (pH and temperature shocks) on mesophilic (37 °C) and thermophilic (55 °C) dark fermentative H₂ production from xylose (50 mM) and, for the first time, on the composition of the active microbial community was evaluated. At 37 °C, an acidic shock (pH 3, 24 h) resulted in the highest yield of 0.8 mol H₂ mol^?1 xylose. The H₂ and butyrate yield correlated with the relative abundance of Clostridiaceae in the mesophilic active microbial community, whereas Lactobacillaceae were the most abundant non-hydrogenic competitors according to RNA-based analysis. At 55 °C, Clostridium and Thermoanaerobacterium were linked to H₂ production, but only an alkaline shock (pH 10, 24 h) repressed lactate production, resulting in the highest yield of 1.2 mol H₂ mol^?1 xylose. This study showed that pretreatments differentially affect the structure and productivity of the active mesophilic and thermophilic microbial community developed from an inoculum.