Effects of initial lactic acid concentration, HRTs, and OLRs on bio-hydrogen production from lactate-type fermentation

A batch test and continuous operation were performed to identify the effect of lactate on hydrogen production at pH 4.5. When the initial lactic acid concentration was increased from 0 to 8 g/L in the batch test, the hydrogen yield also increased from 1.41 to 1.72 mol-H₂/mol-glucose. The system exhibited a long lag time and an insignificant hydrogen yield with 16 g-lactic acid/L. A continuous stirred tank reactor (CSTR) was operated at different organic loading rates (OLRs: 10, 15, 20 and 40 g/L/day) and hydraulic retention times (HRTs: 6, 12 and 24 h). At an OLR of 20 g-glucose/L/day and 12 h of HRT, the hydrogen yield was 1.2 mol-H₂/mol-glucose. The yield decreased with a 24 h HRT. Even though lactate was one of the major constituents of volatile fatty acids (VFAs), hydrogen production was feasible throughout the operation. Clostridium sp. was the dominant hydrogen-producing bacteria in the system.     

Efficient production of butyric acid from Jerusalem artichoke by immobilized Clostridium tyrobutyricum in a fibrous-bed bioreactor

Butyric acid is an important specialty chemical with wide industrial applications. The feasible large-scale fermentation for the economical production of butyric acid requires low-cost substrate and efficient process. In the present study, butyric acid production by immobilized Clostridium tyrobutyricum was successfully performed in a fibrous-bed bioreactor using Jerusalem artichoke as the substrate. Repeated-batch fermentation was carried out to produce butyric acid with a high butyrate yield (0.44 g/g), high productivity (2.75 g/L/h) and a butyrate concentration of 27.5 g/L. Furthermore, fed-batch fermentation using sulfuric acid pretreated Jerusalem artichoke hydrolysate resulted in a high butyric acid concentration of 60.4 g/L, with the yield of 0.38 g/g and the selectivity of ∼85.1 (85.1 g butyric acid/g acetic acid). Thus, the production of butyric acid from Jerusalem artichoke on a commercial scale could be achieved based on the system developed in this work.     

Metabolic and energetic aspects of biohydrogen production of Clostridium tyrobutyricum: The effects of hydraulic retention time and peptone addition

This study evaluates the microbial metabolism and energy demand in fermentative biohydrogen production using Clostridium tyrobutyricum FYa102 at different hydraulic retention times (HRT) over a period of 1-18 h. The hydrogen yield shows a positive correlation with the butyrate yield, the B/A ratio, and the Y_H2/2(Y_HAc+Y_HBu) ratio, but a negative correlation with the lactate yield. A decrease in HRT, which is accompanied by an increased biomass growth, tends to decrease the B/A ratio, due presumably to a higher energy demand for microbial growth. The production of lactate at a low HRT, however, may involve an unfavorable change in e⁻ equiv distribution to result in a reduced hydrogen production. Finally, the relatively high hydrogen yields observed in the bioreactor with the peptone addition may be ascribed to the utilization of peptone as an additional energy and/or amino-acid source, thus reducing the glucose demand for biomass growth during the hydrogen production process.     

Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment

Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, l-alanine, l-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6-19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (P_s) of 7.784 mL and production yield of (P_r) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R² 0.9988. The final pH of the broth was increased from 7.0 to 8.5-8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H₂ evolution rate and higher butyrate concentration in the fermentate.     

Using cheese whey for hydrogen and methane generation in a two-stage continuous process with alternative pH controlling approaches

This study focuses on the exploitation of cheese whey as a source for hydrogen and methane, in a two-stage continuous process. Mesophilic fermentative hydrogen production from undiluted cheese whey was investigated at a hydraulic retention time (HRT) of 24 h. Alkalinity addition (NaHCO₃) or an automatic pH controller were used, to maintain the pH culture at a constant value of 5.2. The hydrogen production rate was 2.9 ± 0.2 L/Lreactor/d, while the yield of hydrogen produced was approximately 0.78 ± 0.05 mol H₂/mol glucose consumed, with alkalinity addition, while the respective values when using pH control were 1.9 ± 0.1 L/Lreactor/d and 0.61 ± 0.04 mol H₂/mol glucose consumed. The corresponding yields of hydrogen produced were 2.9 L of H₂/L cheese whey and 1.9 L of H₂/L cheese whey, respectively. The effluent from the hydrogenogenic reactor was further digested to biogas in a continuous mesophilic anaerobic bioreactor. The anaerobic digester was operated at an HRT of 20d and produced approximately 1 L CH₄/d, corresponding to a yield of 6.7 L CH₄/L of influent. The chemical oxygen demand (COD) elimination reached 95.3% demonstrating that cheese whey could be efficiently used for hydrogen and methane production, in a two-stage process.     

Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01

An unsterile and continuous fermentation process was developed based on a halophilic bacterium termed Halomonas TD01 isolated from a salt lake in Xinjiang, China. The strain reached 80 g/L cell dry weight containing 80% poly(3-hydroxybutyrate) (PHB) on glucose salt medium during a 56 h fed-batch process. In a 14-day open unsterile and continuous process, the cells grew to an average of 40 g/L cell dry weight containing 60% PHB in the first fermentor with glucose salt medium. Continuous pumping of cultures from the first fermentor to the second fermentor containing the nitrogen-deficient glucose salt medium diluted the cells but allowed them to maintain a PHB level of between 65% and 70% of cell dry weight. Glucose to PHB conversions were between 20% and 30% in the first fermentor and above 50% in the second one. This unsterile and continuous fermentation process opens a new area for reducing the cost in polyhydroxyalkanoates production.     

Hydrogen production by Rhodopseudomonas palustris WP 3-5 in a serial photobioreactor fed with hydrogen fermentation effluent

In this study, a lab-scale serial photobioreactor composed of three column reactors was constructed and continuously operated to investigate several parameters influencing photohydrogen production when using the synthetic wastewater and the anaerobic hydrogen fermentation effluents as the influents. The results indicated that better hydrogen production rate was obtained when the serial photobioreactor was operated under cellular recycling at a short HRT of 8 h. The serial photobioreactor maintained high hydrogen content ca. 80% in the produced gas and 0.4× dilution ratio was the suitable ratio for hydrogen production. When the photobioreactor fed with the real wastewater (Effluent 1) containing 100 mg/L NH₄Cl, Column 1 reactor successfully reduced ammonia concentration to about 60 mg/L for cell synthesis, resulting in a steady hydrogen production in the following two column reactors. The average hydrogen production rate was 205 mL-H₂/L/d.     

Treatment of phenolics containing synthetic wastewater in an internal loop airlift bioreactor (ILALR) using indigenous mixed strain of Pseudomonas sp. under continuous mode of operation

The scope of this study is to evaluate the performance of internal loop airlift bioreactor (ILALR) in treating synthetic wastewater containing phenol and m-cresol, in single and multi component systems. The microbe utilized in the process was an indigenous mixed strain of Pseudomonas sp. isolated from a wastewater treatment plant. The reactor was operated at both lower and higher hydraulic retention times (HRTs) i.e., 4.1 and 8.3 h, respectively, by providing an inlet feed flow rate of 5 and 10 mL/min. Shock loading experiments were also performed up to a maximum concentration of 800 mg/L for phenol at 8.3 h HRT and 500 mg/L for m-cresol at 4.1 h HRT. The study showed complete degradation of both phenol and m-cresol, when they were degraded individually at a HRT of 8.3 h. Experiments with both phenol and m-cresol present as mixtures were performed based on the 2² full factorial design of experiments.     

Process development of succinic acid production by Escherichia coli NZN111 using acetate as an aerobic carbon source

Escherichia coli strain NZN111 could convert glucose to succinic acid efficiently in anaerobic conditions after the induction of gluconeogenic carbon sources in aerobic conditions. Acetate shows a strong effect on both yield and productivity of succinic acid. In this study, the fed-batch process of succinic acid production by NZN111 using acetate in a chemically defined medium in the aerobic stage was investigated and developed. Increasing cell density could increase succinic acid with a productivity of 3.97 g/(L h) in the first 8 h of the anaerobic phase with an overall yield of 1.42 mol/mol glucose in a 5 L fermentor. However, there was strong repression from succinic acid in the later anaerobic stage. When succinic acid exceeded 30 g/L, the glucose consumption rate began to drop sharply along with the succinic acid production rate. Supplementation with glucose from 30 to 70 g/L in the anaerobic stage showed little effect on succinic acid production. Acetic acid and pyruvic acid accumulated had no effect on succinic acid formation because of their low concentration. With acetate as the sole carbon source for aerobic cultivation in the following scale-up, 60.09 g/L of succinic acid was produced with a yield of 1.37 mol/mol in a 50 L bioreactor.     

Swine manure fermentation for hydrogen production

Biohydrogen fermentation using liquid swine manure as substrate supplemented with glucose was investigated in this project. Experiments were conducted using a semi-continuously-fed fermenter (8 L in total volume and 4 L in working volume) with varying pHs from 4.7 through 5.9 under controlled temperature (35 ± 1 °C). The hydraulic retention time (HRT) tested include 16, 20, and 24 h; however, in two pH conditions (5.0 and 5.3), an additional HRT of 12 h was also tried. The experimental design combining HRT and pH provided insight on the fermenter performance in terms of hydrogen generation. The results indicated that both HRT and pH had profound influences on fermentative hydrogen productivity. A rising HRT would lead to greater variation in hydrogen concentration in the offgas and the best HRT was found to be 16 h for the fermenter in this study. The best pH value in correspondence to the highest hydrogen generation was revealed to be 5.0 among all the pHs studied. There was no obvious inhibition on hydrogen production by methanogenesis when methane content in the offgas was lower than 2%. Otherwise, an inverse linear relationship between hydrogen and methane content was observed with a correlation coefficient of 0.9699. Therefore, to increase hydrogen content in the offgas, methane production has to be limited to below 2%.     

l-Lactic acid production by Bacillus subtilis MUR1 in continuous culture

A novel UV-induced mutant strain of recombinant Bacillus subtilis MUR1 was used for the production of l-LA in continuous cultures with a variety of culture conditions. The maximal productivity of 17.6 g/L/h was obtained with a l-LA concentration of 44.1 g/L at the dilution rate of 0.4 h⁻¹. The highest concentration of l-LA (77.1 g/L) was produced at the dilution rate of 0.05 h⁻¹. This study showed that the maximum l-LA productivity of B. subtilis MUR1 which can only last for a very short period of time during the exponential phase in fed-batch cultures, can be extended indefinitely at steady state in continuous cultures. l-LA production increased with the increase of yeast extract concentrations in the medium. Moreover, temperature, agitation rate and various glucose concentrations in the feed were compared in continuous cultures. Different nitrogen sources (lysine, glutamine, ammonium sulphate and corn steep liquor) were studied to partly or completely replace yeast extract in the medium, most of them showed positive effects on l-LA production and cell growth. The l-LA productivities from continuous cultures in this study are higher than the productivity of current microbial industrial processes which use Lactobacillus to produce l-LA.     

Hydrogen production by immobilized R. faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria E. harbinense B49

In order to increase the hydrogen yield from glucose, hydrogen production by immobilized Rhodopseudomonas faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria Ethanoligenens harbinense B49 was investigated. The soluble metabolites from dark-fermentation mainly were ethanol and acetate, which could be further utilized for photo-hydrogen production. Hydrogen production by B49 was noticeably affected by the glucose and phosphate buffer concentration. The maximum hydrogen yield (1.83 mol H₂/mol glucose) was obtained at 9 g/l glucose. In addition, we found that the ratio of acetate/ethanol (A/E) increased with increasing phosphate buffer concentration, which is favorable to further photo-hydrogen production. The total hydrogen yield during dark- and photo-fermentation reached its maximum value (6.32 mol H₂/mol glucose) using 9 g/l glucose, 30 mmol/l phosphate buffers and immobilized R. faecalis RLD-53. Results demonstrated that the combination of dark- and photo- fermentation was an effective and efficient process to improve hydrogen yield from a single substrate.     

Hydrolysis of lignocellulosic feedstock by novel cellulases originating from Pseudomonas sp. CL3 for fermentative hydrogen production

A newly isolated indigenous bacterium Pseudomonas sp. CL3 was able to produce novel cellulases consisting of endo-β-1,4-d-glucanase (80 and 100 kDa), exo-β-1,4-d-glucanase (55 kDa) and β-1,4-d-glucosidase (65 kDa) characterized by enzyme assay and zymography analysis. In addition, the CL3 strain also produced xylanase with a molecular weight of 20 kDa. The optimal temperature for enzyme activity was 50, 45, 45 and 55 °C for endo-β-1,4-d-glucanase, exo-β-1,4-d-glucanase, β-1,4-d-glucosidase and xylanase, respectively. All the enzymes displayed optimal activity at pH 6.0. The cellulases/xylanase could hydrolyze cellulosic materials very effectively and were thus used to hydrolyze natural agricultural waste (i.e., bagasse) for clean energy (H₂) production by Clostridiumpasteurianum CH4 using separate hydrolysis and fermentation process. The maximum hydrogen production rate and cumulative hydrogen production were 35 ml/L/h and 1420 ml/L, respectively, with a hydrogen yield of around 0.96 mol H₂/mol glucose.     

Lipid production by Lipomyces starkeyi cells in glucose solution without auxiliary nutrients

Two-stage fermentation process was used for lipid production by Lipomyces starkeyi AS 2.1560 in glucose solution without auxiliary nutrients. In the first stage, cells were cultivated in a nutrient-rich medium for propagation. In the second stage, cells were resuspended in glucose solution to achieve high cellular lipid contents. The effects of the inocula age, cell density and initial glucose concentration on lipid production were briefly studied. When high cell density fermentation was performed in a 7-L stirred-tank bioreactor for 40 h using non-sterile glucose solution as carbon source, the biomass, lipid and lipid content reached 104.6 g/L, 67.9 g/L and 64.9%, respectively. More significantly, lipid productivity reached 2.0 g/L h during the initial 16 h-period and 1.6 g/L h for the entire culture. Our results demonstrated that cell propagation and lipid accumulation processes can be spatially separated, allowing further optimization to improve both processes. The two-stage fermentation method should have a great potential to develop more efficient processes to convert renewable materials into biofuel and related products.     

Sequential production of two biopolymers-levan and poly-ε-lysine by microbial fermentation

Sequential fermentation for the production of two invaluable biopolymers, levan and poly-ε-lysine (ε-PL), has been successfully developed. It involves fermentation of Bacillus subtilis (natto) Takahashi in sucrose medium to produce levan, separation of levan product from small remaining sugar molecules by ultrafiltration and fermentation of the remnant from levan production by Streptomyces albulus to produce ε-PL. In the process, 50-60 g/L of levan was produced (100% recovery after precipitation by ethanol). The remnant from levan production with glucose adjusted to 30 g/L and with combined use of yeast extract (10 g/L), (NH₄)₂SO₄ (2 g/L) and basal salts was proven to be suitable for ε-PL production. 4.37 g/L of ε-PL accumulation (85% recovery after purification) was reached in 72 h using two-stage fermentation with control of pH. The process of using remnant (waste) from levan fermentation for the second biopolymer (ε-PL) production is unprecedented and the products obtained are environmental-friendly.     

Hydrogen and methane production through two-stage mesophilic anaerobic digestion of olive pulp

The present study focused on the anaerobic biohydrogen production from olive pulp (two phase olive mill wastes, TPOMW) and the subsequent anaerobic treatment of the effluent for methane production under mesophilic conditions in a two-stage process. Biohydrogen production from water-diluted (1:4) olive pulp was investigated at hydraulic retention times (HRT) of 30 h, 14.5 h and 7.5 h while methane production from the effluent of hydrogenogenic reactor was studied at 20 d, 15 d, 10 d and 5 d HRT. In comparison with previous studies, it has been shown that the thermophilic hydrogen production process was more efficient than the mesophilic one in both hydrogen production rate and yield. The methanogenic reactor was successfully operated at 20, 15 and 10 days HRT while it failed when an HRT of 5 days was applied. Methane productivity reached the maximum value of 1.13 ± 0.08 L/L/d at 10 days HRT whereas the methane yield increased with the HRT. The Anaerobic Digestion Model no. 1 (ADM1) was applied to the obtained experimental data from the methanogenic reactor to simulate the digester response at all HRT tested. The ability of the model to predict the experimental results was evident even in the case of the process failure, thus implying that the ADM1 could be a valuable tool for process design even in the case of a complex feedstock. In general, the two-stage anaerobic digestion proved to be a stable, reliable and effective process for energy recovery and stabilization treatment of olive pulp.     

Simultaneous production of citric acid and invertase by Yarrowia lipolytica SUC+ transformants

Simultaneous production of citric acid (CA) and invertase by Yarrowia lipolytica A-101-B56-5 (SUC⁺ clone) growing from sucrose, mixture of glucose and fructose, glucose or glycerol was investigated. Among the tested substrates the highest concentration of CA was reached from glycerol (57.15 g/L) with high yield (Y_CA/S = 0.6 g/g). When sucrose was used, comparable amount of CA was secreted (45 g/L) with slightly higher yield (Y_CA/S = 0.643 g/g). In all cultures amount of isocitrate (ICA) was below 2% of total citrates. Considering invertase production, the best carbon source appeared to be sucrose (72 380 U/L). The highest yield of CA and invertase biosynthesis calculated for 1 g of biomass was obtained for cells growing from glycerol (9.9 g/g and 4325 U/g, respectively). Concentrates of extra- and intracellular invertase of the highest activity were obtained from sucrose as substrate (0.5 and 1.8 × 10⁶ U/L, respectively).     

Engineered Escherichia coli capable of co-utilization of cellobiose and xylose

Natural ability to ferment the major sugars (glucose and xylose) of plant biomass is an advantageous feature of Escherichia coli in biofuel production. However, excess glucose completely inhibits xylose utilization in E. coli and decreases yield and productivity of fermentation due to sequential utilization of xylose after glucose. As an approach to overcome this drawback, E. coli MG1655 was engineered for simultaneous glucose (in the form of cellobiose) and xylose utilization by a combination of genetic and evolutionary engineering strategies. The recombinant E. coli was capable of utilizing approximately 6 g/L of cellobiose and 2 g/L of xylose in approximately 36 h, whereas wild-type E. coli was unable to utilize xylose completely in the presence of 6 g/L of glucose even after 75 hours. The engineered strain also co-utilized cellobiose with mannose or galactose; however, it was unable to metabolize cellobiose in the presence of arabinose and glucose. Successful cellobiose and xylose co-fermentation is a vital step for simultaneous saccharification and co-fermentation process and a promising step towards consolidated bioprocessing.     

Improvement of butanol fermentation by supplementation of butyric acid produced from a brown alga

This study investigated butanol fermentation using glucose and culture broth containing butyrate from the butyrate fermentation of a brown alga, Laminaria japonica. Prior to the use of the biologically-produced butyrate, the initial glucose in tryptone-yeast extract acetate (TYA) medium was first optimized for butanol fermentation using Clostridium saccharoperbutylacetonicum N1-4 ATCC 27021^T. Then, a commercially-acquired (synthetic) butyrate was supplemented to the TYA medium containing the optimal glucose concentration (around 30 and 60 g/L). According to the experimental results, the highest butanol carbon yield (0.580 C-mol/C-mol) was obtained from the fermentation of 36.65 g/L glucose and 7.29 g/L synthetic butyrate. Fermentation of a similar amount of glucose (32.28 g/L) in the absence of butyrate gave a butanol carbon yield of 0.402 C-mol/C-mol. For the experiment with fermented butyrate, a 100 g/L biomass of brown alga was fermented by Clostridium tyrobutyricum ATCC 25755 and the culture broth containing butyrate was used to prepare TYA medium after removing the bacterial cells. Fermentation using the synthetic butyrate and the biologically-produced butyrate (4.95 g/L) gave a comparable butanol concentration (13.23 g/L) and butanol carbon yield (0.513 C-mol/C-mol). Overall, this study proved that the addition of fermented butyrate from brown alga fermentation could be an effective way to improve butanol production. Furthermore, the reuse of spent medium and the absence of rigorous purification of the broth containing butyrate would lower the production cost of the fermentation.     

Influence of substrate concentration on the stability and yield of continuous biohydrogen production

The effect of substrate concentration (sucrose) on the stability and yield of a continuous fermentative process producing hydrogen was studied. High substrate concentrations are attractive from an energy standpoint as they would minimise the energy required for heating. The reactor was a CSTR; temperature was maintained at 35 degrees C; pH was controlled between 5.2 and 5.3, and the hydraulic retention time (HRT) was 12 h. Online measurements were taken for ORP, pH, temperature, %CO2, gas output and %H2, and data logged using a MatLAB data acquisition toolbox. Steady-state operation was obtained at 10, 20 and 40 g/L of sucrose in the influent, but a subsequent step change to 50 g/L was unsustainable. The hydrogen content ranged between 50% and 60%. The yield of hydrogen decreased as the substrate concentration increased from 1.7 +/- 0.2 mol/mol hexose added at 10 g/L, to 0.8 +/- 0.1 mol/mol at 50 g/L. Sparging with nitrogen improved the hydrogen yield by at least 35% at 40 g/L and at least 33% at 50 g/L sucrose. Sparging also enabled steady-state operation at 50 g/L sucrose. Addition of an extra 4 g/L of n-butyric acid to the reactor operating at 40 g/L sucrose increased the butyrate concentration from 9,830 to 18,900 mg/L, immediately stopping gas production and initiating the production of propionate, whilst the addition of 2 g/L taking the butyrate concentration to 12,200 mg/L did not do so. It was shown that operation at 50 g/L sucrose in a CSTR in butyrate fermentation is possible.