Effect of pH on biomass production and carbohydrate accumulation of Chlorella vulgaris JSC-6 under autotrophic, mixotrophic, and photoheterotrophic cultivation.

Microalgal biomass, known as the third generation feedstock for biofuels production, is currently being explored mainly for lipids and functional components. However, the potential of microalgal carbohydrates has not been evaluated. In this investigation, Chlorella vulgaris JSC-6 was used for carbohydrates production from CO2 and fatty acids under different cultivation strategies to meet the requirements of a CO2-neutral and clean fermentation system for biofuel production. Autotrophic cultivation resulted in better carbon assimilation and carbohydrate accumulation; about 1.4 g CO2 could be converted to 1 g biomass, of which 50% are carbohydrates. Assimilation of fatty acids in photoheterotrophic and mixotrophic modes was influenced by pH, and pH 7-7.5 supported butyrate and acetate assimilation. The maximum carbohydrate content (49.86%) was attained in mixotrophic mode, and the ratio of the simple sugars glucose-xylose-arabinose was 1:0.11:0.02. The higher glucose content makes the microalgal biomass a suitable feedstock for sugar-based fermentations.

Succinic acid fermentation with immobilized Actinobacillus succinogenes using hydrolysate of carbohydrate-rich microalgal biomass.

This work aimed to study the efficiency of polyvinyl-alcohol-immobilized Actinobacillus succinogenes ATCC55618 for succinic acid (SA) production. Batch fermentation (pH 7, 45% CO2 gas at 0.04 vvm) using glucose (40 g L-1) resulted in SA titer, 26.7 g L-1; productivity, 3.33 g L-1h-1; yield, 0.621 g g-1. Fed-batch mode with cyclic extrication of SA from the medium markedly enhanced the yield to 0.699 g g-1 and concentration to 59.5 g L-1. Batch fermentation using sugars derived from Chlorella vulgaris ESP-31 without yeast extract gave a SA productivity, concentration, and yield of 1.82 g L-1h-1, 36.1 g L-1, and 0.720 g g-1, respectively. Furthermore, continuous fermentation (at 6 h HRT) with microalgal sugar improved the productivity and yield to 3.53 g L-1h-1 and 0.62 g g-1, respectively, which is comparable to those obtained by using glucose. This study reports the highest productivity for SA fermentation using microalgae-derived sugars.

Exploring fermentation strategies for enhanced lactic acid production with polyvinyl alcohol-immobilized Lactobacillus plantarum 23 using microalgae as feedstock.

Lactic acid (LA) fermentation was conducted with suspended and immobilized cells of an isolated Lactobacillus plantarum 23 strain using various fermentation strategies. Glucose and an alternative, relatively inexpensive carbon source - the hydrolysate of microalga Chlorella vulgaris ESP-31, were used as the carbon source. Batch fermentation using immobilized cells of L. plantarum 23 could enhance LA titer and yield by 43% and 39%, respectively, when compared with the suspended culture. Fed-batch culture integrated with in situ LA removal via ion exchange raised LA productivity by 72% by overcoming product inhibition. The highest LA productivity from glucose with PVA immobilized cells was 14.22 g/L/h, achieved under continuous operation at 50% w/v loading of immobilized beads and hydraulic retention time (HRT) of 2 h. PVA immobilized L. plantarum 23 could also use microalgal hydrolysate as the renewable carbon source, and the highest LA productivity was 9.93 g/L/h under continuous fermentation at 4 h HRT.

Dark H2 fermentation from sucrose and xylose using H2-producing indigenous bacteria: feasibility and kinetic studies.

Cellulosic materials are the major components in biomass feedstock used for bioenergy production. Hydrolytic products of cellulosic substances consist primarily of hexose (e.g., glucose) and pentose (e.g., xylose). In this study, the efficiency of fermentative conversion of sucrose (representing hexose) and xylose into H2 was examined with seven H2-producing pure strains isolated from a high-rate H2-producing system in our recent work. The isolates were identified as Clostridium butyricum (strains CGS2 and CGS5), Clostridium pasteurianum (strains CH1, CH4, CH5, and CH7), and Klebsiella sp. Batch H2 fermentation shows that only Cl. butyricum and Klebsiella sp. strains could utilize xylose for H2 production, while all of them can grow and produce H2 on sucrose. Among all strains examined, Cl. butyricum CGS5 was the best H2 producer on xylose with the highest H2 production rate and yield of 212.5 ml/h/l and 0.73 mol H2/mol xylose, respectively, taking place at 20 g COD/l of xylose. In contrast, Cl. pasteurianum CH4 was most efficient in converting sucrose to H2; the highest H2 production rate (569 ml/h/l) and yield (2.07 mol H2/mol hexose) were obtained at a sucrose concentration of 40 g COD/l. The substrate preference of the H2-producing isolates was consistent with the bacterial community structure that existed in the bioreactor, showing that Cl. butyricum and Cl. pasteurianum were predominant in the cultures grown on xylose and sucrose, respectively. Irrespective of the carbon substrate used, butyrate and acetate were the predominant soluble metabolites. Shake-flask cultures displayed higher H2 productivity over static ones, indicating the importance of efficient mass transfer for H2 production. The dependence of cell growth and H2 production on carbon substrate concentration could be described by the proposed kinetic models with good agreements.

Cellulosic hydrogen production with a sequencing bacterial hydrolysis and dark fermentation strategy.

In this study, cellulose hydrolysis activity of two mixed bacterial consortia (NS and QS) was investigated. Combination of NS culture and BHM medium exhibited better hydrolytic activity under the optimal condition of 35 degrees C, initial pH 7.0, and 100rpm agitation. The NS culture could hydrolyze carboxymethyl cellulose (CMC), rice husk, bagasse and filter paper, among which CMC gave the best hydrolysis performance. The CMC hydrolysis efficiency increased with increasing CMC concentration from 5 to 50g/l. With a CMC concentration of 10g/l, the total reducing sugar (RS) production and the RS producing rate reached 5531.0mg/l and 92.9mg/l/h, respectively. Furthermore, seven H2-producing bacterial isolates (mainly Clostridium species) were used to convert the cellulose hydrolysate into H2 energy. With an initial RS concentration of 0.8g/l, the H2 production and yield was approximately 23.8ml/l and 1.21mmol H2/g RS (0.097mmol H2/g cellulose), respectively.

Simultaneous production of 2,3-butanediol, ethanol and hydrogen with a Klebsiella sp. strain isolated from sewage sludge.

A Klebsiella sp. HE1 strain isolated from hydrogen-producing sewage sludge was examined for its ability to produce H2 and other valuable soluble metabolites (e.g., ethanol and 2,3-butanediol) from sucrose-based medium. The effect of pH and carbon substrate concentration on the production of soluble and gaseous products was investigated. The major soluble metabolite produced from Klebsiella sp. HE1 was 2,3-butanediol, accounting for over 42-58% of soluble microbial products (SMP) and its production efficiency enhanced after increasing the initial culture pH to 7.3 (without pH control). The HE1 strain also produced ethanol (contributing to 29-42% of total SMP) and a small amount of lactic acid and acetic acid. The gaseous products consisted of H2 (25-36%) and CO2 (64-75%). The optimal cumulative hydrogen production (2.7 l) and hydrogen yield (0.92mol H2 mol sucrose(-1)) were obtained at an initial sucrose concentration of 30g CODl(-1) (i.e., 26.7gl(-1)), which also led to the highest production rate for H2 (3.26mmol h(-1)l(-1)), ethanol (6.75mmol h(-1)l(-1)) and 2,3-butanediol (7.14mmol h(-1)l(-1)). The highest yield for H2, ethanol and 2,3-butanediol was 0.92, 0.81 and 0.59molmol-sucrose(-1), respectively. As for the overall energy production performance, the highest energy generation rate was 27.7kJ h(-1)l(-1) and the best energy yield was 2.45kJmolsucrose(-1), which was obtained at a sucrose concentration of 30 and 20g CODl(-1), respectively.

Dark hydrogen fermentation from hydrolyzed starch treated with recombinant amylase originating from Caldimonas taiwanensis On1.

Starch is one of the most abundant resources on earth and is suited to serve as a cost-effective feedstock for biological hydrogen production. However, producing hydrogen from direct fermentation of starch is usually inefficient, as the starch hydrolysis is often the rate-limiting step. Therefore, in the present work, enzymatic starch hydrolysis was conducted to enhance the feasibility of using starch feedstock for H2 production. The amylase (with a molecular weight of ca. 112 kDa) used for starch hydrolysis was produced from a recombinant E. coli harboring an amylase gene originating from Caldimonas taiwanensis On1. Using statistical experimental design, the optimal pH and temperature for starch hydrolysis with the recombinant amylase was pH 6.86 and 52.4 degrees C, respectively, at an initial starch concentration of 7 g/L. The hydrolyzed products contained mainly glucose, maltotriose, and maltotetrose, while a tiny amount of maltose was also detected. The enzymatically hydrolyzed products of soluble starch and cassava starch were used as the substrate for dark hydrogen fermentation using Clostridium butyricum CGS2 and Clostridium pasteurianum CH4. The highest H2 production rate (vH2) and yield (YH2) of C. butyricum CGS2 was 124.0 mL/h/L and 6.32 mmol H2/g COD, respectively, both obtained with the hydrolysate of cassava starch. The best H2 production rate (63.0 mL/h/L) of C. pasteurianum CH4 occurred when using hydrolyzed cassava starch as the substrate, whereas the highest yield (9.95 mmol H2/g COD) was obtained with the hydrolyzed soluble starch.

Recovery of gold from industrial wastewater by extracellular proteins obtained from a thermophilic bacterium Tepidimonas fonticaldi AT-A2.

Biosorption has emerged as a promising alternative approach for treating wastewater with dilute metal contents in a green and cost effective way. In this study, extracellular proteins of an isolated thermophilic bacterium (Tepidimonas fonticaldi AT-A2) were used as biosorbent to recover precious metal (i.e., Au) from wastewater. The Au (III) adsorption capacity on the T. fonticaldi AT-A2 proteins was the highest when the pH was set at about 4.0-5.0. The adsorption capacity increased with increasing temperature from 15 to 70°C. Adsorption isotherm studies show that both Langmuir and Freundrich models could describe the adsorption equilibrium. The maximum adsorption capacity of Au (III) at 50°C and pH 5 could reach 9.7mg Au/mg protein. The protein-based biosorbent was also used for the recovery of Au from a wastewater containing 15mg/L of Au, achieving a high adsorption capacity of 1.45mg Au/mg protein and a removal efficiency of 71%.

Cultivation of Chlorella vulgaris JSC-6 with swine wastewater for simultaneous nutrient/COD removal and carbohydrate production.

Swine wastewater, containing a high concentration of COD and ammonia nitrogen, is suitable for the growth of microalgae, leading to simultaneous COD/nutrients removal from the wastewater. In this study, an isolated carbohydrate-rich microalga Chlorella vulgaris JSC-6 was adopted to perform swine wastewater treatment. Nearly 60-70% COD removal and 40-90% NH3-N removal was achieved in the mixotrophic and heterotrophic culture, depending on the dilution ratio of the wastewater, while the highest removal percentage was obtained with 20-fold diluted wastewater. Mixotrophic cultivation by using fivefold diluted wastewater resulted in the highest biomass concentration of 3.96 g/L. The carbohydrate content of the microalga grown on the wastewater can reach up to 58% (per dry weight). The results indicated that the microalgae-based wastewater treatment can efficiently reduce the nutrients and COD level, and the resulting microalgal biomass had high carbohydrate content, thereby having potential applications for the fermentative production of biofuels or chemicals.

Improving protein production of indigenous microalga Chlorella vulgaris FSP-E by photobioreactor design and cultivation strategies.

Fish meal is currently the major protein source for commercial aquaculture feed. Due to its unstable supply and increasing price, fish meal is becoming more expensive and its availability is expected to face significant challenges in the near future. Therefore, feasible alternatives to fish meal are urgently required. Microalgae have been recognized as the most promising candidates to replace fish meal because the protein composition of microalgae is similar to fish meal and the supply of microalgae-based proteins is sustainable. In this study, an indigenous microalga (Chlorella vulgaris FSP-E) with high protein content was selected, and its feasibility as an aquaculture protein source was explored. An innovative photobioreactor (PBR) utilizing cold cathode fluorescent lamps as an internal light source was designed to cultivate the FSP-E strain for protein production. This PBR could achieve a maximum biomass and protein productivity of 699 and 365 mg/L/day, respectively, under an optimum urea and iron concentration of 12.4 mM and 90 µM, respectively. In addition, amino acid analysis of the microalgal protein showed that up to 70% of the proteins in this microalgal strain consist of indispensable amino acids. Thus, C. vulgaris FSP-E appears to be a viable alternative protein source for the aquaculture industry.

Exploring the inhibitory characteristics of acid hydrolysates upon butanol fermentation: A toxicological assessment.

This study aimed to quantitatively evaluate the inhibitor tolerance of butanol-producing bacterium Clostridium acetobutylicum. The inhibitory effect of the inhibitors generated by acid pretreatment of biomass feedstock on butanol fermentation decreased in the order of formic acid>oxalic acid>furfural>5-HMF>Na2SO4. C. acetobutylicum has a small tolerance range for furfural (1.06-2.6g/L) and 5-HMF (1.99-2.3g/L). However, the inhibitory effect of Na2SO4 appears to have a wide range, with a chronic toxicity for C. acetobutylicum. All the results could explain, in quantitative manner, the instability of butanol fermentation with C. acetobutylicum from various acid-pretreated feedstocks caused by the fermentation inhibitors.

Recovery of high-value metals from geothermal sites by biosorption and bioaccumulation.

Generation of geothermal energy is associated with a significant amount of geothermal fluids, which may be abundant in high-value metals, such as lithium, cesium, rubidium, and other precious and rare earth metals. The recovery of high-value metals from geothermal fluids would thus have both economic and environmental benefits. The conventional technologies applied to achieve this are mostly physicochemical, which may be energy intensive, pose the risk of secondary pollution whilst being inefficient in recovering metals from dilute solutions. Biological methods, based on biosorption or bioaccumulation, have recently emerged as alternative approaches, as they are more environmentally friendly, cost effective, and suitable for treating wastewater with dilute metal contents. This article provides a comprehensive review of the related biological technologies used to recover the high-value metals present in geothermal fluids as well as critical discussion on the key issues that are often used to evaluate the effectiveness of those methods.

Perspectives on cultivation strategies and photobioreactor designs for photo-fermentative hydrogen production.

Photosynthetic bacteria have considerable biotechnological potential for biological hydrogen production due to higher substrate conversion efficiency and hydrogen yield. Phototrophic fermentation using photosynthetic bacteria has a major advantage of being able to further convert the byproducts originating from dark fermentation (e.g., volatile fatty acids) to hydrogen. Through the combination of dark and photo-fermentation processes, organic feedstock is fully converted into gaseous product (H2) at the highest possible H2 yield, with significant reduction of chemical oxygen demand (COD). The performance of photo-fermentation is highly dependent on the medium composition, culture conditions, and photobioreactor design. Therefore, this article provides a critical review of the effects of key factors affecting the photo-hydrogen production efficiency of photosynthetic bacteria, and also summarizes the strategies being applied in promoting the performance of photo-fermentation.

Using a starch-rich mutant of Arabidopsis thaliana as feedstock for fermentative hydrogen production.

A mutant plant (Arabidopsis thaliana), sex1-1 (starch excess 1-1), accumulating high starch content in leaves was created to serve as better biomass feedstock for a H2-producing strain Clostridium butyricum CGS2, which efficiently utilizes starch for H2 production but cannot assimilate cellulosic materials. The starch content of the mutant plant increased to 10.67 mg/fresh weight, which is four times higher than that of wild type plant. Using sex1-1 mutant plant as feedstock, C. butyricum CGS2 could produce 490.4 ml/l of H2 with a H2 production rate of 32.9 ml/h/l. The H2 production performance appeared to increase with the increase in the concentration of mutant plant from 2.5 to 10 g/l. The highest H2 to plant biomass yield was nearly 49 ml/g for the mutant plant. This study successfully demonstrated the feasibility of using a starch-rich mutant plant for more effective bioH2 production with C. butyricum CGS2.

Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1.

A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and ß-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H2 production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H2 production rate and yield of 57.7 ml/h/L and 2.03 mol H2/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H2 fermentation.

Biohydrogen production from lignocellulosic feedstock.

Due to the recent energy crisis and rising concern over climate change, the development of clean alternative energy sources is of significant interest. Biohydrogen produced from cellulosic feedstock, such as second generation feedstock (lignocellulosic biomass) and third generation feedstock (carbohydrate-rich microalgae), is a promising candidate as a clean, CO2-neutral, non-polluting and high efficiency energy carrier to meet the future needs. This article reviews state-of-the-art technology on lignocellulosic biohydrogen production in terms of feedstock pretreatment, saccharification strategy, and fermentation technology. Future developments of integrated biohydrogen processes leading to efficient waste reduction, low CO2 emission and high overall hydrogen yield is discussed.

Multicomponent cellulase production by Cellulomonas biazotea NCIM-2550 and its applications for cellulosic biohydrogen production.

Among four cellulolytic microorganisms examined, Cellulomonas biazotea NCIM-2550 can grow on various cellulosic substrates and produce reducing sugar. The activity of cellulases (endoglucanase, exoglucanase, and cellobiase), xylanase, amylase, and lignin class of enzymes produced by C. biazotea was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (carboxymethyl cellulose [CMC], sugarcane bagasse [SCB], and xylan) used for growth. Effects of physicochemical conditions on cellulolytic enzyme production were systematically investigated. Using MnCl(2) as a metal additive significantly induces the cellulase enzyme system, resulting in more reducing sugar production. The efficiency of fermentative conversion of the hydrolyzed SCB and xylan into clean H(2) energy was examined with seven H(2)-producing pure bacterial isolates. Only Clostridiumbutyricum CGS5 exhibited efficient H(2) production performance with the hydrolysate of SCB and xylan. The cumulative H(2) production and H(2) yield from using bagasse hydrolysate (initial reducing sugar concentration = 1.545 g/L) were approximately 72.61 mL/L and 2.13 mmol H(2)/g reducing sugar (or 1.91 mmol H(2)/g cellulose), respectively. Using xylan hydrolysate (initial reducing sugar concentration = 0.345 g/L) as substrate could also attain a cumulative H(2) production and H(2) yield of 87.02 mL/L and 5.03 mmol H(2)/g reducing sugar (or 4.01 mmol H(2)/g cellulose), respectively.

Dark fermentative hydrogen production from enzymatic hydrolysate of xylan and pretreated rice straw by Clostridium butyricum CGS5.

Xylan and rice straw were used to produce H(2) via a two-stage approach combining feedstock pretreatment/hydrolysis and dark H(2) fermentation. Acinetobacter junii F6-02 was used to produce cellulolytic enzymes (mainly xylanase) to hydrolyze xylan and pretreated rice straw. The hydrolysates were converted to H(2) by Clostridium butyricum CGS5 via dark fermentation. Investigation of kinetics of xylanase on xylan and NaOH-pretreated rice straw shows nu(max) values of 8.6 and 3.6g/L/h, and K(m) values of 10.6 and 26.9 g/L, respectively. A maximum hydrogen production rate of 62.5 and 26.8 ml/h/L was obtained from hydrolysate of xylan and pretreated rice straw, respectively, while the hydrogen yield was 0.70 and 0.76 mol H(2)/mol xylose, respectively. Simultaneous saccharification and BioH(2) fermentation from xylan was also conducted but giving a lower hydrogen production rate (35.3 ml/h/L) than that of the two-stage process.

Strategies to enhance cell growth and achieve high-level oil production of a Chlorella vulgaris isolate.

The autotrophic growth of an oil-rich indigenous microalgal isolate, identified as Chlorella vulgaris C--C, was promoted by using engineering strategies to obtain the microalgal oil for biodiesel synthesis. Illumination with a light/dark cycle of 14/10 (i.e., 14 h light-on and 10 h light-off) resulted in a high overall oil production rate (v(oil)) of 9.78 mg/L/day and a high electricity conversion efficiency (E(c)) of 23.7 mg cell/kw h. When using a NaHCO(3) concentration of 1,500 mg/L as carbon source, the v(oil) and E(c) were maximal at 100 mg/L/day and 128 mg/kw h, respectively. A Monod type model was used to describe the microalgal growth kinetics with an estimated maximum specific growth rate (mu(max)) of 0.605 day(-1) and a half saturation coefficient (K(s)) of 124.9 mg/L. An optimal nitrogen source (KNO(3)) concentration of 625 mg/L could further enhance the microalgal biomass and oil production, leading to a nearly 6.19 fold increase in v(oil) value.

Bioreactors configured with distributors and carriers enhance the performance of continuous dark hydrogen fermentation.

Anaerobic granular sludge bed (AnGSB) bioreactors were supplemented with activated carbon carriers and combined with distributors (e.g., acrylic resin board, stainless steel net and plastic net) installed at different locations to investigate the effect of distributor/carrier on biohydrogen production efficiency. The results show that plastic net stimulated the substrate/microorganisms contact and sludge granulation, thereby leading to a much better H(2) production performance when compared with those obtained from traditional CSTR. The highest H(2) production rate (7.89 L/h/L) and yield (3.42 mol H(2)/mol sucrose) were obtained when two pieces of plastic nets were installed at both 4 cm and 16 cm from the bottom of AnGSB without carrier addition and the bioreactor was operated at a HRT of 0.5h. For the AnGSB installed with two pieces of plastic net distributors, addition of carriers led to significant improvement on the H(2) production efficiency at a longer HRT (1-4h) when compared with the carrier-absent system.