Optimal fermentation conditions for maximizing the ethanol production by Kluyveromyces fragilis from cheese whey powder

Cheese whey powder (CWP) is an attractive raw material for ethanol production since it is a dried and concentrated form of CW and contains lactose in addition to nitrogen, phosphate and other essential nutrients. In the present work, deproteinized CWP was utilized as fermentation medium for ethanol production by Kluyveromyces fragilis. The individual and combined effects of initial lactose concentration (50-150 kg m⁻³), temperature (25-35 °C) and inoculum concentration (1-3 kg m⁻³) were investigated through a 2³ full-factorial central composite design, and the optimal conditions for maximizing the ethanol production were determined. According to the statistical analysis, in the studied range of values, only the initial lactose concentration had a significant effect on ethanol production, resulting in higher product formation as the initial substrate concentration was increased. Assays with initial lactose concentration varying from 150 to 250 kg m⁻³ were thus performed and revealed that the use of 200 kg m⁻³ initial lactose concentration, inoculum concentration of 1 kg m⁻³ and temperature of 35 °C were the best conditions for maximizing the ethanol production from CWP solution. Under these conditions, 80.95 kg m⁻³ of ethanol was obtained after 44 h of fermentation.     

Continuous fermentative hydrogen production from cheese whey wastewater under thermophilic anaerobic conditions

Hydrogen (H₂) production from cheese processing wastewater via dark anaerobic fermentation was conducted using mixed microbial communities under thermophilic conditions. The effects of varying hydraulic retention time (HRT: 1, 2 and 3.5 days) and especially high organic load rates (OLR: 21, 35 and 47 g chemical oxygen demand (COD)/l/day) on biohydrogen production in a continuous stirred tank reactor were investigated. The biogas contained 5–82% (45% on average) hydrogen and the hydrogen production rate ranged from 0.3 to 7.9 l H₂/l/day (2.5 l/l/day on average). H₂ yields of 22, 15 and 5 mmol/g COD (at a constant influent COD of 40 g/l) were achieved at HRT values of 3.5, 2, and 1 days, respectively. On the other hand, H₂ yields were monitored to be 3, 9 and 6 mmol/g COD, for OLR values of 47, 35 and 21 g COD/l/day, when HRT was kept constant at 1 day. The total measurable volatile fatty acid concentration in the effluent (as a function of influent COD) ranged between 118 and 27,012 mg/l, which was mainly composed of acetic acid, iso-butyric acid, butyric acid, propionic acid, formate and lactate. Ethanol and acetone production was also monitored from time to time.To characterize the microbial community in the bioreactor at different HRTs, DNA in mixed liquor samples was extracted immediately for PCR amplification of 16S RNA gene using eubacterial primers corresponding to 8F and 518R. The PCR product was cloned and subjected to DNA sequencing. The sequencing results were analyzed by using MegaBlast available on NCBI website which showed 99% identity to uncultured Thermoanaerobacteriaceae bacterium.     

Feasibility of biohydrogen production from cheese whey using a UASB reactor: Links between microbial community and reactor performance

The present study examines the feasibility of producing hydrogen by dark fermentation using unsterilised cheese whey in a UASB reactor. A lab-scale UASB reactor was operated for more than 250 days and unsterilised whey was used as the feed. The evolution of the microbial community was studied during reactor operation using molecular biology tools (T-RFLP, 16S rRNA cloning library and FISH) and conventional microbiological techniques.     

Thermophilic anaerobic digestion of cheese whey: Coupling H2 and CH4 production

The thermophilic anaerobic digestion of cheese whey was evaluated using a single and two stage configuration (H₂–CH₄) in a sequencing batch reactor (SBR). The single stage process presented stable performance with a specific methane production (SMP) of 314.5 ± 6.6 L CH₄ kg⁻¹ COD_feed (Chemical oxygen demand) at a hydraulic retention time (HRT) of 8.3 days. On the contrary, the two stage process presented instabilities at an HRT of 12.5 days with acid accumulation being observed in the methanogenesis phase at an earlier stage. This behaviour was indicative of process inhibition by high concentrations of sodium and potassium ions as a consequence of pH control during the H₂ producing stage. In spite of this phenomenon, this condition attained the highest SMP value (340.4 ± 40 L CH₄ kg⁻¹ COD_feed). The performance of the anaerobic digestion process was also analysed by means of Fourier transform infrared (FTIR) and ¹H nuclear magnetic resonance (¹H NMR) spectroscopy. The two stage process showed higher content in triacylglycerol groups probably associated with changes in archaeal lipid complexes as a microbial response to a higher salinity environment.     

Biohydrogen production from dark fermentation of cheese whey: Influence of pH

Hydrogen production from cheese whey through dark fermentation was investigated in this study in order to systematically analyse the effects of the operating pH. The effluents from pecorino cheese and mozzarella cheese production were the substrates used for the fermentation tests. Either CW only or a mixture of CW and heat-shocked activated sludge were used in mesophilic pH-controlled batch fermentation experiments. The results indicated that hydrogen production was strongly affected by multiple factors including the substrate characteristics, the addition of an inoculum as well as the pH. The process variables were found to affect to a varying extent numerous interrelated aspects of the fermentation process, including the hydrogen production potential, the type of fermentation pathways, as well as the process kinetics. The fermentation products varied largely with the operating conditions and mirrored the H₂ yield. Significant fermentative biohydrogen production was attained at pHs of 6.5–7.5, with the best performance in terms of H₂ generation potential (171.3 NL H₂/kg TOC) being observed for CW from mozzarella cheese production, at a pH value of 6.0 with the heat-shocked inoculum.     

Optimization and modelling of dark fermentative hydrogen production from cheese whey by Enterobacter aerogenes 2822

In India, annually about 3.3–5 million tons of cheese whey is produced which may causes serious problems for the environment if left untreated. In this study, pretreated cheese whey was utilized to produce hydrogen via dark fermentation by Enterobacter aerogenes 2822 cells in 2 L double walled cylindrical bioreactor having working volume of 1.5 L. Effect of change in total carbohydrate concentration in cheese whey (CW_TC, 20–45 g L^?1), temperature (T, 25–37 °C) and pH (5.5–7.5) was investigated on volumetric hydrogen production rate (VHPR) using Box Behnken design (BBD). Experimental VHPR of 24.7 mL L^?1 h^?1 was attained at an optimum concentration of 32.5 g L^?1 CW_TC, 31 °C T and 6.5 pH, which was in good correlation with predicted rate of 23.2 mL L^?1 h^?1. Mathematical models based on Monod and logistic equations were developed to describe the kinetics of substrate consumption and growth profile of E. aerogenes 2822 under optimum conditions. While for the modelling of fermentative hydrogen production in batch mode, Modified Gompertz equation and Leudeking-Piret models were used which gave proper simulated fitting. These results will add significant values to cheese whey by converting it into a clean form of bioenergy.     

Simultaneous hydrogen gas formation and COD removal from cheese whey wastewater by electrohydrolysis

Cheese whey (CW) was subjected to DC voltages between 0.5 and 5 V for hydrogen gas production with simultaneous COD removal by electrohydrolysis of CW organics. Hydrogen gas formation and COD removal were investigated at different DC voltages using aluminum electrodes. The highest cumulative hydrogen production (5551 mL), hydrogen yield (1709 mL H₂ g⁻¹ COD), hydrogen gas formation rate (913 ml d⁻¹), and percent hydrogen (99%) in the gas phase were obtained with 5 V DC voltage within 158 h. Energy conversion efficiency reached the highest level (80.7%) at 3 V DC voltage with cumulative hydrogen production of 4808 mL and hydrogen yield of 1366 mL H₂ g⁻¹ COD. Hydrogen gas was mainly produced by electrohydrolysis of CW organics due to low H₂ gas production in water and CW control experiments. The highest COD removal (22%) was also obtained with 3 V DC voltage. Major COD removal mechanism was anaerobic degradation of carbohydrates producing volatile fatty acids (VFA) and CO₂. Hydrogen gas was produced by reaction of protons released from VFAs and electrons provided by DC current. Hydrogen gas production by electrohydrolysis of CW solution was proven to be an effective method with simultaneous COD removal.     

Electro-hydrolysis of cheese whey solution for hydrogen gas production and chemical oxygen demand (COD) removal using photo-voltaic cells (PVC)

Diluted cheese whey (CW) solution was used for hydrogen gas production by electro-hydrolysis using photo-voltaic cells (PVC) as source of electricity. Effects of initial chemical oxygen demand (COD) concentration on the rate and yield of hydrogen gas production were investigated using a completely mixed and sealed reactor with aluminum electrodes. Cumulative hydrogen gas formation (CHF) increased with increasing initial COD concentration. The highest cumulative hydrogen gas volume (26472 mL), hydrogen gas production rate (4553 mL d⁻¹), hydrogen yield (7004 mL H₂ g⁻¹ COD), and percent COD removal (21.5%) were obtained with initial COD of 35172 mg L⁻¹. H₂ gas formation from water control was only 5365 mL. pH of the CW solution increased with decreasing conductivities during the course of experiments. Gas phase contained more than 99% H₂ at the end of experiments. The highest energy efficiency (20.4%) was also obtained with the highest COD content. Nearly pure hydrogen gas formation by electro-hydrolysis of cheese whey using PVC panels was proven to be an effective method.     

Kinetic modelling of continuous production of ethanol from cheese whey

A kinetic model for continuous fermentation of ethanol from cheese whey was developed. The model accounts for substrate limitation, substrate inhibition, ethanol inhibition and cell death. Three bioreactors of 5 L volume each were operated at different hydraulic retention times (HRT) ranging from 18 to 42 h and initial lactose concentrations ranging from 50 to 150 g/L. The experimental data were used to validate the model. The model predicted the cell, lactose and ethanol concentrations with high accuracy (R² = 0.96–0.99). The cell concentration, lactose utilization and ethanol production were significantly affected by hydraulic retention time and initial substrate concentration. Lactose utilizations of 98, 91 and 83% were obtained with 50, 100 and 150 g/L initial lactose concentrations at 42 h HRT. The highest cell concentration (5.5 g/L), highest ethanol concentration (58.0 g/L) and maximum ethanol yield (99.6% of theoretical) were achieved at 42 h HRT and 150 g/L initial lactose concentration.     

Direction of glucose fermentation towards hydrogen or ethanol production through on-line pH control

The present study investigated the production of hydrogen (H₂) and ethanol from glucose in an Anaerobic Continuous Stirred Tank Reactor (ACSTR). Effects of hydraulic retention time (HRT) and pH on the preference of producing H₂ and/or ethanol and other soluble metabolic products in an open anaerobic enriched culture were studied. Production rates of H₂ and ethanol increased with the increase of biomass concentration. Open anaerobic fermentation was directed and managed through on-line pH control for the production of H₂ or ethanol. Hydrogen was produced by ethanol and acetate-butyrate type fermentations. pH has strong effect on the H₂ or ethanol production by changing fermentation pathways. ACSTR produced mainly ethanol at over pH 5.5 whereas highest H₂ production was obtained at pH 5.0. pH 4.9 favored the lactate production and accumulation of lactate inhibited the biomass concentration in the reactor and the production of H₂ and ethanol. The microbial community structure quickly responded to pH changes and the Clostridia dominated in ACSTR during the study. H₂ production was maintained mainly by Clostridium butyricum whereas in the presence of Bacillus coagulans glucose oxidation was directed to lactate production.     

Continuous hydrogen production from cofermentation of sugarcane vinasse and cheese whey in a thermophilic anaerobic fluidized bed reactor

The co-fermentation of vinasse and cheese whey (CW) was evaluated in this study by using two thermophilic (55° C) anaerobic fluidized bed reactors (AFBRs). In AFBR using vinasse and CW (AFBR-V-CW), the CW was added in increasing proportions (2, 4, 6, 8, and 10 g COD.L^?1) to vinasse (10 g COD.L^?1) to assess the advantage of adding CW to vinasse. By decreasing the hydraulic retention time (HRT) from 8 h to 1 h in AFBR-V, maximum hydrogen yield (HY), production rate (HPR), and H₂ content (H₂%) of 1.01 ± 0.06 mmol H₂.g COD^?1, 2.54 ± 0.39 L H₂.d^?1.L^?1, and 47.3 ± 2.9%, respectively, were observed at an HRT of 6 h. The increase in CW concentration to values over 2 g COD.L^?1 in AFBR-V-CW decreased the HY, PVH, and H₂%, with observed maximum values of 0.82 ± 0.07 mmol H₂.g COD^?1, 1.41 ± 0.24 L H₂.d^?1.L^?1, and 55.5 ± 3.7%, respectively, at an HRT of 8 h. The comparison of AFBR-V-CW and AFBR-V showed that the co-fermentation of vinasse with 2 g COD.L^?1 of CW increased the HPR, H₂%, and HY by 117%, 68%, and 82%, respectively.     

Performance comparison among different multifunctional reactors operated under energy self-sufficiency for sustainable hydrogen production from ethanol

Four ethanol-derived hydrogen production processes including conventional ethanol steam reforming (ESR), sorption enhanced steam reforming (SESR), chemical looping reforming (CLR) and sorption enhanced chemical looping reforming (SECLR) were simulated on the basis of energy self-sufficiency, i.e. process energy requirement supplied by burning some of the produced hydrogen. The process performances in terms of hydrogen productivity, hydrogen purity, ethanol conversion, CO₂ capture ability and thermal efficiency were compared at their maximized net hydrogen. The simulation results showed that the sorption enhanced processes yield better performances than the conventional ESR and CLR because their in situ CO₂ sorption increases hydrogen production and provides heat from the sorption reaction. SECLR is the most promising process as it offers the highest net hydrogen with high-purity hydrogen at low energy requirement. Only 12.5% of the produced hydrogen was diverted into combustion to fulfill the process's energy requirement. The thermal efficiency of SECLR was evaluated at 86% at its optimal condition.     

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.     

Enhancing photo fermentative hydrogen production using ethanol rich dark fermentation effluents

The present study demonstrates the feasibility of a two-phase biorefinery process applied to waste substrates producing ethanol rich effluents. The process includes a dark fermentation step followed by photo fermentation and it is able to optimize hydrogen production from waste biomass. The study was conducted using winery wastewater as feedstock. The results indicate that no additional treatments are required when an appropriate dilution of the initial waste is applied. Microbial consortia contained in the winery wastewater promoted a fermentative ethanol pathway. The ethanol rich effluent was converted into hydrogen by phototrophic microorganisms. Despite the presence of inhibiting compounds, the adoption of a mixed phototrophic culture allowed to obtain good results in terms of hydrogen production. Specifically, up to 310 mLH₂ gCOD_consumed^?1 were obtained in the photo fermentative stage. The effectiveness of ethanol rich dark fermentation effluents for hydrogen production enhancement was demonstrated. Noteworthy, polyhydroxybutyrate was also produced during the experiments. The work faces two of the major challenges in the sequential dark fermentation and photo fermentation technology applied to real waste substrates: the minimization of pre-treatments and the enhancement of the hydrogen production yields using ethanol rich DFEs.     

Different start-up strategies to enhance biohydrogen production from cheese whey in UASB reactors

The effect of different operational strategies and inoculum structure (granules and disaggregated granules) during the start-up of four up-flow anaerobic sludge blanket hydrogenogenic reactors was investigated. The more stable volumetric hydrogen production rates obtained were 0.38 and 0.36 L H₂/L-d, in reactors operated with a constant organic loading rate (OLR) with both inoculum structures, whereas in reactors operated with an increasing OLR methane started to be produced earliest in time. Specific hydrogenogenic activity results proved that the disaggregated inoculum produced a more active biomass than the granular one, but not granule formation was evident. The methane hydrogenotrophic activity was the main limitation of the systems evaluated. In the reactors inoculated with disaggregated sludge the start-up strategy did not influence the bacterial DGGE fingerprint, in contrast to the reactors started-up with granular sludge; members of the Clostridium genus were always present. The results demonstrated that operational conditions during the start-up period are crucial for the production of hydrogenogenic biomass.     

Ni-hydrocalumite derived catalysts for ethanol steam reforming on hydrogen production

Hydrocalumite derived catalysts prepared by co-precipitation with non-noble metal Nickel(Ni) as main active site were tested in ethanol steam reforming, and the influences of Ni (5,10,15 wt%) content were mainly tested in this research. Meanwhile, the physicochemical properties of the prepared catalysts were analyzed through different characterizations including BET, X-ray diffraction (XRD), H₂-temperature programmed reduction (H₂-TPR) and CO₂-temperature programmed desorption (TPD). As the Ni increased, the specific surface area, crystallite size of Ni, reducibility and basicity of catalysts were changed, which further affected their activities. On this basis, the best performance in this catalytic system was presented when Ni in the catalysts was 15 wt%, the ethanol conversion and hydrogen yield could reach almost 100% and 85% at 650 °C respectively. Thus, this kind of catalyst is effective for ethanol steam reforming.     

Effect of molar ratio of water / ethanol on hydrogen selectivity in catalytic production of hydrogen using steam reforming of ethanol

As fossil energy resources are shrinking, the increase in global energy needs and environmental pollution paved the way to the search for new and renewable energy resources. Therefore, the future of energy technology is being built on the use of hydrogen, which is one of the cleanest and most efficient renewable energy sources, and steam reforming is becoming the utmost method to produce hydrogen. This study focuses on the operation condition of steam reforming of ethanol on catalyst materials, which were shaped using active metals such as Ni, Cu and Cs and supporting materials which were activated by carbon and LiAlO₂. These catalyst materials were tested to produce hydrogen gas using different water/ethanol mole ratio at different temperatures and a constant feed flow rate. The evaluation regarding hydrogen selectivity results and the percentage of hydrogen in the products revealed that NiCuCs/LiAlO₂ catalyst showed the highest performance at all water/ethanol ratios and temperatures between 300 and 600 °C.     

Large capacity hydrogen production by microwave discharge plasma in liquid fuels ethanol

In situ hydrogen production technologies have attracted attentions because of hydrogen storage and transportation safety issues. Discharge plasma technology for hydrogen production is of fast response, large capacity, small scale and portability, which is suitable for automobiles and ships. In this paper, a method for producing hydrogen by microwave discharge in ethanol solution was introduced. A microwave discharge reactor of direct standing wave coupling (MDRSWC) was designed, which was suitable for on-board hydrogen production. The characteristics of large capacity hydrogen production by applying MDRSWC in liquid ethanol were investigated. Depending on the experimental conditions of ethanol concentration and microwave power, the flow rate of hydrogen production was achieved ranging from 28.93 to 72.48 g/h. In addition to main hydrogen and carbon dioxide, a small amount of methane and acetylene as by-products were detected. By optimizing the experimental conditions, the experimental results showed that the flow rate of hydrogen, the percentage concentration of hydrogen and the energy yield of hydrogen production were 72.48 g/h, 58.1% and 48.32 g/kWh respectively. This work could provide a potentially effective hydrogen production method for on-board hydrogen utilization device.     

Possibility of hydrogen production during cheese whey fermentation process by different strains of psychrophilic bacteria

The aim of this study was to determine the possibility of applying psychrophilic bacteria for hydrogen production in whey biofermentation process. Experiments were conducted in 500-mL anaerobic respirometers at a temperature of 20 °C. The initial organic load of fermentation tanks reached 10 g COD/L. Depending on the experimental variant, analyses were carried out for psychrophilic bacteria isolated from underground water and from demersal lake water that represented Gammaproteobacteria class – Rahnella aquatilis (9 strains) and Firmicutes phylum: Carnobacterium maltaromaticum, Trichococcus collinsii and Clostridium algidixylanolyticum. The effectiveness of biogas production was diversified and strain-specific, ranging from 126.48 to 4737.72 mL/g bacterial biomass. The highest concentration of H₂ in biogas, ranging from 65.15% to 69.12% and effectiveness of H₂ production from 1587.47 to 3087.57 mL/g bacterial biomass, were determined for R. aquatilis strains isolated from the demersal lake water. The lowest H₂ concentration in the gaseous metabolites, i.e. 15.46% to 20.70%, was noted for bacteria of the phylum Firmicutes.