Dilute-acid pretreatment of barley straw for biological hydrogen production using Caldicellulosiruptor saccharolyticus

The main objective of this study was to use the fermentability test to investigate the feasibility of applying various dilute acids in the pretreatment of barley straw for biological hydrogen production. At a fixed acid loading of 1% (w/w dry matter) 28–32% of barley straw was converted to soluble monomeric sugars, while at a fixed combined severity of −0.8 30–32% of the straw was converted to soluble monomeric sugars. With fermentability tests at sugar concentrations 10 and 20 g/L the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed good hydrogen production on hydrolysates of straw pretreated with H₃PO₄ and H₂SO₄, and to a lesser extent, HNO₃. The fermentability of the hydrolysate of straw pretreated with HCl was lower compared to the other acids but equally high as that of pure sugars. At sugar concentration 30 g/L the fermentability of all hydrolysates was low.     

Lipid production from sweet sorghum bagasse through yeast fermentation

Cryptococcus curvatus has great potential in fermenting unconditioned hydrolysates of sweet sorghum bagasse. With hydrolysates obtained by enzymatic hydrolysis of the solid pretreated by microwave with lime, the maximal yeast cell dry weight and lipid content were 10.83 g/l and 73.26%, respectively. For hydrolysates obtained in the same way but without lime, these two parameters were 15.50 g/l and 63.98%, respectively. During yeast fermentation, glucose and xylose were consumed simultaneously while cellobiose was released from the residual bagasse. The presence of lime, on one hand, made cellulose more accessible to enzymes as evidenced by higher total reducing sugar release compared to that without during enzymatic hydrolysis step; on the other hand, it caused the degradation of sugars to non-sugar chemicals during pretreatment step. As a result, higher lipid yield of 0.11 g/g bagasse or 0.65 ton/hectare of land was achieved from the pathway of microwave pretreatment and enzymatic hydrolysis while 0.09 g/g bagasse or 0.51 ton/hectare of land was attained from the process of lime-assisted microwave pretreatment followed by the same enzymatic saccharification.     

Growth and hydrogen production characteristics of Caldicellulosiruptor saccharolyticus on chemically defined minimal media

Caldicellulosiruptor saccharolyticus is an extreme thermophilic bacterium recognized for its saccharolytic ability and superior ability to produce high yields of hydrogen. However, most studies have been made using yeast extract (YE) as a rich but expensive nutrient source. For the first time, we show that C. saccharolyticus is able to grow on defined minimal media, including essential vitamins, provided that CO₂ was allowed to accumulate sufficiently in the culture broth to activate growth. Growth and hydrogen production performance on minimal media was analyzed in both batch and continuous mode. Absence of YE resulted in similar or higher hydrogen yields and specific hydrogen productivities but lower volumetric hydrogen productivities than with YE. The results also indicate that YE is used as a carbon- and energy source thus affecting metabolic flux calculations. This study clarified that YE is not essential making C. saccharolyticus more attractive for fundamental studies on its metabolism and future industrial exploitation.     

Biohydrogen production from wheat straw hydrolysate using Caldicellulosiruptor saccharolyticus followed by biogas production in a two-step uncoupled process

A two-step, un-coupled process producing hydrogen (H₂) from wheat straw using Caldicellulosiruptor saccharolyticus in a ‘Continuously stirred tank reactor’ (CSTR) followed by anaerobic digestion of its effluent to produce methane (CH₄) was investigated. C. saccharolyticus was able to convert wheat straw hydrolysate to hydrogen at maximum production rate of approximately 5.2 L H₂/L/Day. The organic compounds in the effluent collected from the CSTR were successfully converted to CH₄ through anaerobic digestion performed in an ‘Up-flow anaerobic sludge bioreactor’ (UASB) reactor at a maximum production rate of 2.6 L CH₄/L/day. The maximum energy output of the process (10.9 kJ/g of straw) was about 57% of the total energy, and 67% of the energy contributed by the sugar fraction, contained in the wheat straw. Sparging the hydrogenogenic CSTR with the flue gas of the UASB reactor ((60% v/v) CH₄ and (40% v/v) CO₂) decreased the H₂ production rate by 44%, which was due to the significant presence of CO₂. The presence of CH₄ alone, like N₂, was indifferent to growth and H₂ production by C. saccharolyticus. Hence, sparging with upgraded CH₄ would guarantee successful hydrogen production from lignocellulosic biomass prior to anaerobic digestion and thus, reasonably high conversion efficiency can be achieved.     

Evaluation of the influence of CO2 on hydrogen production by Caldicellulosiruptor saccharolyticus

Stripping gas is generally used to improve hydrogen yields in fermentations. Since CO₂ is relatively easy to separate from hydrogen it could be an interesting stripping gas. However, a higher partial CO₂ pressure is accompanied with an increased CO₂ uptake in the liquid, where it hydrolyses and induces an increased requirement of NaOH to maintain the pH. This enhances the osmotic pressure in the culture by 30%, which inhibited the growth of Caldicellulosiruptor saccharolyticus. Indications for this conclusion are: i) Inhibition could almost completely be circumvented by reducing the bicarbonate through decreasing the pH (from 6.5 to 5.5), ii) Growth rates were reduced by 60 ± 10% at an osmotic pressure of 0.218 ± 0.005 osm/kg H₂O independently of the stripping gas, iii) Increased extracellular DNA and protein concentrations were observed as a function of the osmotic pressure. In addition to growth inhibition, the increased sodium bicarbonate in the effluent will contribute to a negative environmental impact when applied at industrial scale.     

Application of sweet sorghum for biodiesel production by heterotrophic microalga Chlorella protothecoides

Microalga Chlorella protothecoides can grow heterotrophically with glucose as the carbon source and accumulate high proportion of lipids. The microalgal lipids are suitable for biodiesel production. To further increase lipid yield and reduce biodiesel cost, sweet sorghum juice was investigated as an alternative carbon source to glucose in the present study. When the initial reducing sugar concentration was 10 g L⁻¹ in the culture medium, the dry cell yield and lipid content were 5.1 g L⁻¹ and 52.5% using enzymatic hydrolyzates of sweet sorghum juice as the carbon source after 120 h-culture in flasks. The lipid yield was 35.7% higher than that using glucose. When 3.0 g L⁻¹ yeast extract was added to the medium, the dry cell yield and lipid productivity was increased to 1.2 g L⁻¹ day⁻¹ and 586.8 mg L⁻¹ day⁻¹. Biodiesel produced from the lipid of C. protothecoides through acid catalyzed transesterification was analyzed by GC–MS, and the three most abundant components were oleic acid methyl ester, cetane acid methyl ester and linoleic acid methyl ester. The results indicate that sweet sorghum juice could effectively enhance algal lipid production, and its application may reduce the cost of algae-based biodiesel.     

Effect of medium composition on biohydrogen production by the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus

Up to now, the analysis of the effects of medium composition on biohydrogen production of Caldicellulosiruptor saccharolyticus was focused mainly on salt concentrations and complex compounds. Within this work we studied the effects of the presence of organic and/or inorganic nitrogen in the medium composition aiming to induce metabolic changes in C. saccharolyticus   to improve its hydrogen evolution rate (HER) and hydrogen specific productivity (qH₂)$\left(q_{{\text{H}}_2}\right)$. Biohydrogen productivities and hydrogen to substrate yield (Y₍H_2/s₎)$\left(Y_{\left({\text{H}}_2/\text{s}\right)}\right)$ of C. saccharolyticus   on xylose in batch mode were higher working in a complex medium than in a defined one; but no significant difference could be settled according to hydrogen to carbon dioxide yields (Y₍H_2/CO₂₎)$\left(Y_{\left({\text{H}}_2/{\text{CO}}_2\right)}\right)$. The specific growth rate of C. saccharolyticus on complex medium was settled at 0.1 h⁻¹ operating in chemostat mode to achieve the highest H₂-productivities under stable conditions. In chemostat mode on xylose, a reduction of the ammonium feed concentration in a defined medium until N-limiting conditions involved higher qH₂$q_{{\text{H}}_2}$ comparing with a straight C-limiting growth.     

Thermophilic biohydrogen production from the enzymatic hydrolysate of cellulose fraction of sweet sorghum bagasse by Thermoanaerobacterium thermosaccharolyticum KKU19: Optimization of media composition

The composition of media for thermophilic biohydrogen production from the enzymatic hydrolysate of cellulose fraction of sweet sorghum bagasse by Thermoanaerobacterium thermosaccharolyticum KKU19 were optimized in order to maximize the hydrogen production potential (P_s). Results from Plackett-Burman design indicated that FeSO₄, CaCl₂, NaHCO₃, and MgCl₂ had a significantly effect (P ≤ 0.05) on P_s. The optimum media composition obtained from the response surface methodology (RSM) with central composite design (CCD), using the hydrolysate at a total sugar concentration of 8.98 g/L, were (all in mg/L): FeSO₄, 1454.65; MgCl_2, 511.36; CaCl_2, 278.62; and NaHCO_3, 2186.41 in which the P_s of 2397 mL H₂/L were obtained. Verification experiment using the optimum media composition in a continuous stirred tank reactor indicated a highly reproducible result in which the P_s of 2608 mL H₂/L was achieved at a hydraulic retention time of 32 h. The results demonstrated that the media composition obtained from the batch experiment using RSM with CCD can be practically applied to continuously produce hydrogen from the hydrolysate with the least error.     

Step-change flow rate liquid hot water pretreatment of sweet sorghum bagasse for enhancement of total sugars recovery

A step-change flow rate liquid hot water (SCFLHW) process was developed with the objective of improving the total sugars recovery including xylose and glucose from sweet sorghum bagasse (SSB). Total xylose yield increased from 60% for batch system (4.25% w/v, 18 min, 184 °C) to about 80% at an initial flow rate of 20 ml/min for 8 min, followed by a rate of 10 ml/min for 10 min. It was hypothesized that the flowing water could enhance the mass transfer and improves the sugars recovery. Although little lignin was removed, the 72 h enzymatic digestibility of treated sample was enhanced by the increase of flow rate. The lignin droplet redeposited on the surface of residual solids might play a crucial role in determining the enzymatic digestibility. The total recovery of sugars from SSB, after the pretreatment (first with the flow rate of 20 ml/min for 8 min, then 10 ml/min for 10 min) and 72 h enzymatic digestion, reached 83.7%, which is superior to the recovery using the SO₂-steam pretreatment or ammonia fiber expansion.     

Batch-based enzymatic saccharification of sweet sorghum bagasse

To improve enzymatic digestibility and sugar concentration, sweet sorghum bagasse was pretreated with alkali and liquid hot water, and then subjected to fed-batch enzymatic hydrolysis. Scanning electron microscopy assay suggested that different pretreatment methods resulted in different composition and structure of residues; these changes had a significant influence on cellulose hydrolysis. Fresh substrate was pretreated and then added at different amounts during the first 48 h to yield a final dry matter content of 30% (w/v). For liquid hot water pretreatment, a maximal glucose concentration of 95.71 g/L, corresponding to 52.85% xylan removal, was obtained with the sweet sorghum bagasse pretreated at 184°C for 18 min. NaOH soaking at ambient conditions removed lignin up to 60%, and the subsequent hydrolysis with cellulase loading of less than 10 FPU/g DM, and substrate supplementation every few hours yield the high glucose and xylose concentrations of 114.89L and 29.93 g/L, respectively after 144 h.     

Application of a modified Anaerobic Digestion Model 1 version for fermentative hydrogen production from sweet sorghum extract by Ruminococcus albus

The aim of the present study was to evaluate the effectiveness of a developed, ADM1-based kinetic model for the hydrogen production process in batch and continuous cultures of the bacterium Ruminococcus albus grown on sweet sorghum extract as the sole carbon source. Although sorghum extract is known to contain at least two different sugars, i.e. sucrose and glucose, no biphasic growth was observed in batch cultures as such growth is reported to occur in cultures of R. albus with mixed substrates. Thus, taking into account that the main sugar of sweet sorghum extract is sucrose, batch experiments with different initial concentrations of sucrose were performed in order to estimate the growth kinetics of the bacterium on this substrate. The kinetic parameters used, concerning the endogenous metabolism of the bacterium as well as those concerning the effect of pH and hydrogen partial pressure (P_H2), were the same as those estimated in a previous study with glucose as carbon source. Subsequently, the experimental data of batch and continuous experiments with sweet sorghum extract were simulated based on the already developed, modified ADM1 model accounting for the use of sugar-based substrate. It was shown that the model which was developed on synthetic substrates was successful in adequately describing the behavior of the microorganism on a real substrate such as sweet sorghum extract and predicting the experimental results quite well with a deviation of the model predictions from the experimental results being between 5-18% for the hydrogen yield.     

Modeling of fermentative hydrogen production from sweet sorghum extract based on modified ADM1

The Anaerobic digestion model 1 (ADM1) framework can be used to predict fermentative hydrogen production, since the latter is directly related to the acidogenic stage of the anaerobic digestion process. In this study, the ADM1 model framework was used to simulate and predict the process of fermentative hydrogen production from the extractable sugars of sweet sorghum biomass. Kinetic parameters for sugars’ consumption and yield coefficients of acetic, propionic and butyric acid production were estimated using the experimental data obtained from the steady states of a CSTR. Batch experiments were used for kinetic parameter validation. Since the ADM1 does not account for metabolic products such as lactic acid and ethanol that are crucial during the fermentative hydrogen production process, the structure of the model was modified to include lactate and ethanol among the metabolites and to improve the predictions. The modified ADM1 simulated satisfactorily batch experiments although further modifications could be made in order to further improve the predictions for the hydrogenogenic process.     

Influence of pH on fermentative hydrogen production from sweet sorghum extract

The present study focused on the influence of pH on the fermentative hydrogen production from the sugars of sweet sorghum extract, in a continuous stirred tank bioreactor. The reactor was operated at a Hydraulic Retention Time of 12 h and a pH range of 3.5–6.5. The maximum hydrogen production rate and yield were obtained at pH 5.3 and were 1752 ± 54 mL H₂/d or 3.50 ± 0.07 L H₂/L reactor/d and 0.93 ± 0.03 mol H₂/mol glucose consumed or 10.51 L H₂/kg sweet sorghum, respectively. The main metabolic product at this pH value was butyric acid. The hydrogen productivity and yield were still at high levels for the pH range of 5.3–4.7, suggesting a pH value of 4.7 as optimum for hydrogen production from an economical point of view, since the energy demand for chemicals is lower at this pH. At this pH range, the dominant fermentation product was butyric acid but when the pH culture sharply decreased to 3.5, hydrogen evolution ceased and the dominant metabolic products were lactic acid and ethanol.     

Effect of Saccharomyces cerevisiae and Zymomonas mobilis on the co-fermentation of sweet sorghum bagasse hydrolysates pretreated under varying conditions

Hydrolysates from sweet sorghum bagasse pretreatment normally contains hexose and pentose sugars, and this complex mixture of sugars presents a challenge for a single microorganism to effectively ferment all sugars to ethanol. In this study, synergistic effects on the co-fermentation of the hydrolysates using Sacchromyces cerevisiae and Zymomonas mobilis ATCC31825 at different ratios were studied. An inoculum of mixed cultures (1:3 and 5:10 g/L, Z. mobilis to S. cerevisiae ratios) was investigated. Each mixed culture was added to the hydrolysates at pH 4.8 and incubated 32 °C for 24 h. The mixture of Z. mobilis to S. cerevisiae at 5:10 g/L showed the highest synergistic effect with ethanol yields of 0.5 g/g. Since the yield for co-culture was significantly higher than the sum of yields from each microorganism, the improvements can be directly related to co-fermentation of hydrolysate by S. cerevisiae and Z. mobilis.     

Pretreatment of Miscanthus for hydrogen production by Thermotoga elfii

Pretreatment methods for the production of fermentable substrates from Miscanthus, a lignocellulosic biomass, were investigated. Results demonstrated an inverse relationship between lignin content and the efficiency of enzymatic hydrolysis of polysaccharides. High delignification values were obtained by the combination of mechanical, i.e. extrusion or milling, and chemical pretreatment (sodium hydroxide). An optimized process consisted of a one-step extrusion-NaOH pretreatment at moderate temperature (70°C). A mass balance of this process in combination with enzymatic hydrolysis showed the following: pretreatment resulted in 77% delignification, a cellulose yield of more than 95% and 44% hydrolysis of hemicellulose. After enzymatic hydrolysis 69% and 38% of the initial cellulose and hemicellulose fraction, respectively, was converted into glucose, xylose and arabinose. Of the initial biomass, 33% was converted into monosaccharides. Normal growth of Thermotoga elfii on hydrolysate was observed and high amounts of hydrogen were produced.     

Effect of substrate concentration on fermentative hydrogen production from sweet sorghum extract

The aim of the present study was to assess the influence of substrate concentration on the fermentative hydrogen production from sweet sorghum extract, in a continuous stirred tank bioreactor. The reactor was operated at a Hydraulic Retention Time (HRT) of 12 h and carbohydrate concentrations ranging from 9.89 to 20.99 g/L, in glucose equivalents. The maximum hydrogen production rate and yield were obtained at the concentration of 17.50 g carbohydrates/L and were 2.93 ± 0.09 L H₂/L reactor/d and 0.74 ± 0.02 mol H₂/mol glucose consumed, corresponding to 8.81 ± 0.02 L H₂/kg sweet sorghum, respectively. The main metabolic product at all steady states was butyric acid, while ethanol production was high at high substrate concentrations. The experiments showed that hydrogen productivity depends significantly on the initial carbohydrate concentration, which also influences the distribution of the metabolic products.     

Coproduction of hydrogen and volatile fatty acid via thermophilic fermentation of sweet sorghum stalk from co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum

This study was focused on investigating the potential of hydrogen and volatile fatty acid (VFA) coproduction. Sweet sorghum stalks (SS) were used as substrate along with Clostridium thermocellum and Clostridium thermosaccharolyticum as production microbes. Inoculation ratio of C. thermosaccharolyticum to C. thermocellum (0:1–1.5:1 and 1:0 v/v), substrate concentrations (2.5–15.0 g/L) and inoculation time intervals of C. thermosaccharolyticum followed by C. thermocellum (0–48 h) were investigated. Experimental data showed that higher yields of hydrogen and VFA were obtained in the co-culture than their individual cultures. The optimum conditions for the highest yield of products found as 1:1 inoculation ratio of both strains, 24 h of time gap between C. thermosaccharolyticum followed by C. thermocellum after the first inoculation and 5 g/L of substrate concentration. The maximum yield of products was observed as hydrogen (5.1 mmol/g-substrate), acetic acid (1.27 g/L) and butyric acid (1.05 g/L) at optimum conditions. The results suggest that SS can be used for simultaneous production of hydrogen and VFA employing co-culture of C. thermocellum and C. thermosaccharolyticum strains. This approach can contribute to the sustainability of biorefinery.     

A quantitative analysis of hydrogen production efficiency of the extreme thermophile Caldicellulosiruptor owensensis OL

Caldicellulosiruptor owensensis strain OL^T (DSM 13100) is an obligately anaerobic, extreme thermophilic bacterium that is capable of utilizing a broad range of carbohydrates and producing H₂ as a metabolic by-product. The performance of C. owensensis on glucose and xylose was analyzed in lab-scale bioreactors to assess its potential use in biohydrogen production. Acetate, H₂, and CO₂ were the main end products during exponential growth of the organism on either sugar. Lactate production was triggered during the transition into the stationary phase and was associated with an increase in the levels of specific l-lactate dehydrogenase activity. In addition, minor amounts of ethanol and propionate could be detected. H₂ and acetate yields were lower on xylose than on glucose, marking an opposite trend to biomass and lactate yields. The influence of elevated H₂ partial pressure on product distribution was more dramatic in xylose-fermenting cultures. Replacement of yeast extract in the medium with a standard vitamins solution improved H₂ yield on both sugars, where it reached 100% of the theoretical maximum, i.e. 4 mol per mol hexose, on glucose. By using the defined medium, both the maximum specific growth rate and the maximum volumetric H₂ production rate of C. owensensis increased significantly on glucose and almost doubled on xylose. Screening other sugars besides glucose and xylose revealed a clear sugar-dependent product-distribution pattern and a direct correlation between biomass and lactate yields, which might be explained considering energy metabolism of the cells. The organism is proposed as a new candidate for biohydrogen production at high yields.     

The effect of nutrient limitation on hydrogen production by batch cultures of Escherichia coli

In order to understand some limiting factors in microbial hydrogen fermentation we have examined hydrogen production by different strains of Escherichia coli grown in batch cultures under different limiting nutrient regimes. The effect of mutations in uptake hydrogenases, in lactate dehydrogenase (ldhA), and fhlA, coding for the regulator of formate hydrogen lyase (fhl) component synthesis, were studied. Each mutation contributed to a modest increase in hydrogen evolution and the effects were synergistic. Various elements were used as limiting nutrient. In batch experiments, limitation for sulfate was without great effect. There was some affect of limiting phosphate with yields approaching 1 mol per mol of glucose. However, strains showed the highest yield of hydrogen per glucose (∼2$\sim 2$) when cultured at limiting concentrations of either ammonia or glucose.