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.     

Biohydrogen production from beet molasses by sequential dark and photofermentation

Biological hydrogen production using renewable resources is a promising possibility to generate hydrogen in a sustainable way. In this study, a sequential dark and photofermentation has been employed for biohydrogen production using sugar beet molasses as a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup⁻ mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NH₄⁺ and yeast extract on sucrose consumption and hydrogen production was determined. The highest hydrogen yield (4.2 mol of H₂/mol sucrose) and maximum volumetric productivity (7.1 mmol H₂/L_c.h) were obtained in the absence of NH₄⁺. The effluent of the dark fermentation containing no NH₄⁺ was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, iron-citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H₂/L_c.h) were attained using the hup⁻ mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H₂/mol sucrose in dark fermentation to 13.7 mol H₂/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H₂/mole of sucrose) by sequential dark and photofermentation.     

Photofermentative hydrogen production using dark fermentation effluent of sugar beet thick juice in outdoor conditions

In the present study, photofermentative hydrogen production on thermophilic dark fermentation effluent (DFE) of sugar beet thick juice was investigated in a solar fed-batch panel photobioreactor (PBR) using Rhodobacter capsulatus YO3 (hup⁻) during summer 2009 in Ankara, Turkey. The DFE was obtained by continuous dark fermentation of sugar beet thick juice by extreme thermophile Caldicellulosiruptor saccharolyticus and it contains acetate (125 mM) and NH₄⁺ (7.7 mM) as the main carbon and nitrogen sources, respectively. The photofermentation process was done in a 4 L plexiglas panel PBR which was daily fed at a rate of 10% of the PBR volume. The DFE was diluted 3 times to adjust the acetate concentration to approximately 40 mM and supplemented with potassium phosphate buffer, Fe and Mo. In order to control the temperature, cooling was provided by recirculating chilled water through a tubing inside the reactor. Hydrogen productivity of 1.12 mmol/L_c/h and molar yield of 77% of theoretical maximum over consumed substrate were attained over 15 days of operation. The results indicated that Rb. capsulatus YO3 could effectively utilize the DFE of sugar beet thick juice for growth and hydrogen production, therefore facilitating the integration of the dark and photo-fermentation processes for sustainable biohydrogen production.     

Biohydrogen production from acid hydrolyzed wastewater treatment sludge by dark fermentation

Waste generation, waste management, sustainable energy production, and global warming are interrelated environmental issues to be considered together. Wastewater treatment sludge is an organic substance rich waste which causes significant environmental problems. However, these wastes can be used as raw material in biofuel generation. This study was designed to investigate the possible utilization of waste sludge in biohydrogen production by taking these facts into consideration. For this purpose, the sludge was first pre-treated with acid and then, the solid (sludge) and liquid (filtrate) phases of acid pre-treated sludge were used as the substrates for biohydrogen generation dark fermentation. Two-factor factorial experimental design method was used in acid hydrolysis of sludge to determine the effect of pH (pH = 2–6) and reaction period (time, min) elution of chemical oxygen demand (COD), total organic carbon (TOC) and total sugar (TS), NH₄N and PO₄P. Statistical evaluation of the results indicated that pH significantly affects the elution of organic carbon and nutrient content of sludge while the reaction time is significant for only organic carbon content. The optimum pretreatment conditions for maximum organic and nutrient elution were determined as pH = 2 and t = 1440 min. The pretreated products, named as filtrate sludge and sludge, conducted to dark fermentation under mesophilic conditions for biohydrogen generation showed that pretreatment of waste sludge at pH = 6 is the best condition giving the maximum yields (Y_H2) as Y_H2 = 24 mmol g⁻¹ Total Sugar _consumed and Y_H2 = 41 mmol g⁻¹ Total sugar consumed, for filtrate and sludge, respectively.     

Bioconversion of sugar industry by-products—molasses and sugar beet pulp for single cell protein production by yeasts

In the bioconversion studies of molasses and sugarbeet pulp to single cell protein by four Candida spp. (utilis, tropicalis, parapsilosis and solani) maximum protein content of 37.5 and 43.4% was achieved from the two substrates, respectively, in 48 h. Candida utilis and C. tropicalis performed better than the other yeasts. The maximum bioconversion efficiency for molasses (43%) was given by C. parapsilosis and for beet pulp (46%) by C. tropicalis in 48 h batch flask fermentations. The bioconversion of beet pulp under controlled conditions was studied using C. tropicalis in a 51 fermentor, which gave 29 and 48% product recovery with 39 and 25% protein level, in a two- and one-stage process, respectively. The one-stage process (simultaneous saccharification and fermentation) was also run in a larger volume and gave 50% product recovery with 29% protein content. The results are discussed in terms of biomass yield, protein content and bioconversion efficiency of yeasts under each condition.     

Biohydrogen production from biomass and industrial wastes by dark fermentation

Hydrogen is a clean energy carrier which has a great potential to be an alternative fuel. Abundant biomass from various industries could be a source for biohydrogen production where combination of waste treatment and energy production would be an advantage. This article summarizes the dark fermentative biohydrogen production from biomass. Types of potential biomass that could be the source for biohydrogen generation such as food and starch-based wastes, cellulosic materials, dairy wastes, palm oil mill effluent and glycerol are discussed in this article. Moreover, the microorganisms, factors affecting biohydrogen production such as undissociated acid, hydrogen partial pressure and metal ions are also discussed.     

Biohydrogen production from cheese processing wastewater by anaerobic fermentation using mixed microbial communities

Hydrogen (H₂) production from simulated cheese processing wastewater via anaerobic fermentation was conducted using mixed microbial communities under mesophilic conditions. In batch H₂ fermentation experiments H₂ yields of 8 and 10 mM/g COD fed were achieved at food-to-microorganism (F/M)$(F/M)$ ratios of 1.0 and 1.5, respectively. Butyric, acetic, propionic, and valeric acids were the major volatile fatty acids (VFA) produced in the fermentation process. Continuous H₂ fermentation experiments were also performed using a completely mixed reactor (CSTR). The pH of the bioreactor was controlled in a range of 4.0–5.0 by addition of carbonate in the feed material. Maximum H₂ yields were between 1.8 and 2.3 mM/g COD fed for the loading rates (LRs) tested with a hydraulic retention time (HRT) of 24 h. Occasionally CH₄ was produced in the biogas with concurrent reductions in H₂ production; however, continuous H₂ production was achieved for over 3 weeks at each LR. The 16S rDNA analysis of DNA extracted from the bioreactors during periods of high H₂ production revealed that more than 50% of the bacteria present were members of the genus Lactobacillus and about 5% were Clostridia. When H₂ production in the bioreactors decreased concurrent reductions in the genus Lactobacillus were also observed. Therefore, the microbial populations in the bioreactors were closely related to the conditions and performance of the bioreactors.     

Biohydrogen production from pretreated sludge and synthetic and real biodiesel wastewater by dark fermentation

Different types of sludge pretreatments were tested, with thermal shock at 90°C to 95°C for 60 minutes plus a 6‐hour rest period achieving the best results for inhibition of methanogen microorganisms and inoculum enrichment with H₂‐producing bacteria, which produced a H₂‐rich biogas (up to 65% mol/mol) without the presence of CH₄. Wastewater from biodiesel production (WBP), containing mainly methanol (128 g/L) and glycerol (4 g/L), was evaluated as a potential substrate to produce H₂ through dark fermentation. Both methanol‐based solutions and methanol‐rich wastewater were not suitable for hydrogen production; however, these effluents showed a strong potential for CH₄‐rich biogas production. A fractional factorial design was employed to evaluate the effect of six substrate‐related variables (glycerol content, 25% and 75%; COD content, 4 and 50 g/L, COD:VSS ratio, 1:1 and 5:1; COD:N:P ratio, 350:0:0 and 350:5:1; NaCl content, 0.5 and 12.0 g/L; and pH, 4.0 and 5.5) on the H₂ production from glycerol‐methanol–based synthetic solutions (synthetic WBP). Some substrate‐related variables had a crucial impact on the hydrogen production potential from WBP, which was significantly affected by the COD and salinity content in the substrate. WBP containing high glycerol (representing until 75% of the COD) and salinity (up to 12 g/L as NaCl) content could be turned into a potential substrate for H₂ production through dark fermentation as long as specific fermentation conditions are maintained, such as pH 5.5 to 5.7 and a substrate COD content up to 50 g/L. Using this condition, glycerol conversion, H₂ productivity, and H₂ yield of 81.3 ± 8.9%, 102.8 ± 18.2 mL H₂/L.d, and 24.5 ± 4.4 mL H₂/g COD_applied, respectively, were obtained.     

Biological hydrogen production from sugar beet molasses by agar immobilized R. capsulatus in a panel photobioreactor

Biohydrogen production from sugar beet molasses was investigated by using agar immobilized R. capsulatus YO3. A panel photobioreactor (1.4 L) was employed for a long-term hydrogen production in both indoor and outdoor conditions. The impact of several initial molasses concentrations on hydrogen production, yield and productivity were assessed. Indoor studies revealed that initial sucrose concentration in molasses should be kept below 20 mM to prevent inhibition of hydrogen production. The highest hydrogen productivity of 0.64 ± 0.06 mmol H₂ L^?1 h^?1 and yield of 12.2 ± 1.5 mol H₂/mol sucrose were obtained in indoors throughout 20 days of operation. For outdoors, hydrogen production continued for 40 days including consecutive 10 rounds under natural outdoor conditions. In outdoor conditions, the maximum hydrogen productivity and yield were 0.79 ± 0.04 mmol H₂ L^?1 h^?1 and 5.2 ± 0.4 mol H₂/mol sucrose respectively. These results indicate that the proposed system is promising for biohydrogen production from molasses at large-scale natural conditions.     

Biohydrogen production by dark fermentation: Experiences of continuous operation in large lab scale

Hydrogen is a clean energy carrier which can be used as fuel in fuel cells. Today, hydrogen is produced mainly by steam reforming of fossil fuels like natural gas or oil. But only hydrogen produced by renewable sources can be called clean energy production. One possibility for hydrogen production is the biological fermentation of biogenous wastes by hydrogen producing bacteria. For the experimental setup four 30-L-working-volume reactors were constructed for continuous biohydrogen production. As inoculum, heat-treated sludge of a wastewater treatment plant was used. Different hydraulic retention times (HRT) were tested and an organic loading rate (OLR) of 2–14 kg VS/m³*d. As starting substrate, waste sugar medium was used. The pH and other parameters were observed to find boundary conditions for a stable continuous process with a minimum of online-control measurements. The high concentration of organic acids in the reactor led to a very low pH, which was controlled manually and online > 4 up to 5.5, otherwise the biohydrogen production decreased rapidly. The gas amount varied with the different OLRs, but could be stabilised on a high level as well as the hydrogen concentration in the gas with 44–52%. No methane was detected in the gas. It turned out, that continuous biohydrogen production with stable gas amounts and qualities could be achieved at different operation conditions. The results showed, that the operation of a continuous biohydrogen reactor has to be observed very carefully to ensure a constant gas production, and that pH-control is necessary to ensure stable operation conditions.     

Bioethanol production from grape and sugar beet pomaces by solid-state fermentation

A suitable alternative to replace fossil fuels is the production of bioethanol from agroindustrial waste. Grape pomace is the most abundant residue in San Juan and sugar beet pomace could be important in the region. Solid-State Fermentation (SSF) is a technology that allows transforming agroindustrial waste into many valuable bioproducts, like ethanol. This work reports a laboratory scale SSF to obtain alcohol from grape and sugar beet pomace by means of Saccharomyces cerevisiae yeasts. The initial conditions of the culture medium were: sugars 16.5% (p/p); pH 4.5; humidity 68% (p/p). Cultures were inoculated with 10⁸ cells/g of pomace, and incubated in anaerobic environment, at 28 °C, during 96 h. SSF showed ethanol maximum concentrations at 48 h and ethanol yield on sugars consumed was more than 82%. Yield attained creates expectation about the use of SSF to obtain fuel alcohol.     

Biohydrogen production from soluble condensed molasses fermentation using anaerobic fermentation

Using anaerobic micro-organisms to convert organic waste to produce hydrogen gas gives the benefits of energy recovery and environmental protection. The objective of this study was to develop a biohydrogen production technology from food wastewater focusing on hydrogen production efficiency and micro-flora community at different hydraulic retention times. Soluble condensed molasses fermentation (CMS) was used as the substrate because it is sacchariferous and ideal for hydrogen production. CMS contains nutrient components that are necessary for bacterial growth: microbial protein, amino acids, organic acids, vitamins and coenzymes. The seed sludge was obtained from the waste activated sludge from a municipal sewage treatment plant in Central Taiwan. This seed sludge was rich in Clostridium sp.A CSTR (continuously stirred tank reactor) lab-scale hydrogen fermentor (working volume, 4.0 L) was operated at a hydraulic retention time (HRT) of 3–24 h with an influent CMS concentration of 40 g COD/L. The results showed that the peak hydrogen production rate of 390 mmol H₂/L-d occurred at an organic loading rate (OLR) of 320 g COD/L-d at a HRT of 3 h. The peak hydrogen yield was obtained at an OLR of 80 g COD/L-d at a HRT of 12 h. At HRT 8 h, all hydrogenase mRNA detected were from Clostridium acetobutylicum-like and Clostridium pasteurianum-like hydrogen-producing bacteria by RT-PCR analysis. RNA based hydrogenase gene and 16S rRNA gene analysis suggests that Clostridium exists in the fermentative hydrogen-producing system and might be the dominant hydrogen-producing bacteria at tested HRTs (except 3 h). The hydrogen production feedstock from CMS is lower than that of sucrose and starch because CMS is a waste and has zero cost, requiring no added nutrients. Therefore, producing hydrogen from food wastewater is a more commercially feasible bioprocess.     

Biohydrogen production from poplar leaves pretreated by different methods using anaerobic mixed bacteria

Leaves are one of the main by-products of forestry. In this study, batch experiments were carried out to convert poplar leaves pretreated by different methods into hydrogen using anaerobic mixed bacteria at 35 °C. The effects of acid (HCl), alkaline (NaOH) and enzymatic (Viscozyme L, a mixture of arabanase, cellulase, β-glucanase, hemicellulase and xylanase) pretreatments on the saccharification of poplar leaves were studied. Furthermore, the effects of acid and enzymatic pretreatment on hydrogen production, together with their corresponding degradation efficiencies for the total reducing sugar (TRS) and metabolites were compared. A maximum cumulative hydrogen yield of 44.92 mL/g-dry poplar leaves was achieved from substrate pretreated with 2% Vicozyme L, which was approximately 3-fold greater than that in raw substrate and 1.34-fold greater than that from substrate pretreated with 4% HCl. The results show that enzymatic pretreatment is an effective method for enhancing the hydrogen yield from poplar leaves.     

Valorization of sugar beet molasses for the production of biohydrogen and 5-aminolevulinic acid by Rhodobacter sphaeroides O.U.001 in a biorefinery concept

The production of biohydrogen and 5-aminolevulinic acid (5-ALA) from sugar beet molasses was investigated within a biorefinery framework. A purple non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides O.U.001, was used for this purpose. The suitability of the molasses for biohydrogen and 5-ALA production was assessed in certain aspects and then five different culture media with various sugar contents (3 g/L, 7 g/L, 14 g/L, 21 g/L and 28 g/L) were prepared. Results have shown that molasses is a promising substrate for the production of biohydrogen and 5-ALA in a biorefinery concept and increasing sugar content results in enhanced product accumulation. Specifically, the highest amount of biohydrogen and 5-ALA was observed in 28 g/L sugar-containing medium (1.01 L H₂/L culture, 23,337 μM). In conclusion, this paper presents the new findings about the enhanced accumulation of biohydrogen and 5-ALA within a biorefinery context.     

Optimized model of fermentable sugar production from Napier grass for biohydrogen generation via dark fermentation

Biohydrogen is a fossil-fuel alternative. Lignocellulosic biomass is a complex part of cellulose-to-simple sugar production. Napier grass, one of the lignocellulosic biomasses, is best for biofuels or biochemicals. The dark fermentation process of Napier grass for biohydrogen proved both cost-effective and environmentally friendly. This grass contains cellulose, hemicellulose and lignin were 35.44 ± 2.01, 20.05 ± 1.55, and 28.473 ± 1.34, respectively. Sodium hydroxide was used in different concentrations to delignify lignocellulose and improve grass glucose recovery. Fermentative hydrogen production from grass biomass processing by microflora was optimized in terms of pH (4.5–7.0) and mesophilic condition (35 ± 2 °C). In this study, mesophilic conditions favored maximum hydrogen production (763.34 ml), indicating that pH 5.5 was suitable for dark-fermentative hydrogen production; study results showed Napier grass could be used successfully for dark fermentation to produce biohydrogen.     

Modelling and prediction of bioethanol production from intermediates and byproduct of sugar beet processing using neural networks

The aim of this work was to model and predict the process of bioethanol production from intermediates and byproduct of sugar beet processing by applying artificial neural networks. Prediction of one substrate fermentation by neural networks had the same input variables (fermentation time and starting sugar content) and one output value (ethanol content, yeast cell number or sugar content). Results showed that a good prediction model could be obtained by networks with single hidden layer. The neural network configuration that gave the best prediction for raw or thin juice fermentation was one with 8 neurons in hidden layer for all observed outputs. On the other side, the optimal number of neurons in hidden layer was found to be 9 and 10 for thick juice and molasses, respectively. Further, all substrates data were merged, which led to introducing an additional input (substrate type) and defining all outputs optimal network architecture to 3-12-1. From the results the conclusion was that artificial neural networks are a good prediction tool for the selected network outputs. Also, these predictive capabilities allowed the application of the Garson's equation for estimating the contribution of selected process parameters on the defined outputs with satisfactory accuracy.     

Biohydrogen production from starch wastewater and application in fuel cell

Biohydrogen was produced from starch in wastewater by anaerobic fermentation. The effects of parameters, such as pH, starch concentration were investigated and optimum operating conditions were determined. The optimal pH and starch concentration for hydrogen production at 37 °C were 6.5 and 5 g/L, respectively with a maximum hydrogen yield of 186 ml/g-starch. The produced biogas contains 99% of hydrogen after passing through KOH solution to remove CO₂. The anaerobic fermentation installation was integrated with a proton-exchange-membrane fuel cell (PEMFC) system for on-line electricity generation. This combination system of biohydrogen and fuel cell achieved a power output of 0.428 W at 0.65 V per cell.     

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.