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.

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.

Characterization of a heat-tolerant Chlorella sp. GD mutant with enhanced photosynthetic CO2 fixation efficiency and its implication as lactic acid fermentation feedstock.

BACKGROUND: Fermentative production of lactic acid from algae-based carbohydrates devoid of lignin has attracted great attention for its potential as a suitable alternative substrate compared to lignocellulosic biomass. RESULTS: A Chlorella sp. GD mutant with enhanced thermo-tolerance was obtained by mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine to overcome outdoor high-temperature inhibition and it was used as a feedstock for fermentative lactic acid production. The indoor experiments showed that biomass, reducing sugar content, photosynthetic O2 evolution rate, photosystem II activity (Fv/Fm and Fv'/Fm'), and chlorophyll content increased as temperature, light intensity, and CO2 concentration increased. The mutant showed similar DIC affinity and initial slope of photosynthetic light response curve (α) as that of the wild type but had higher dissolved inorganic carbon (DIC) utilization capacity and maximum photosynthesis rate (Pmax). Moreover, the PSII activity (Fv'/Fm') in the mutant remained normal without acclimation process after being transferred to photobioreactor. This suggests that efficient utilization of incident high light and enhanced carbon fixation with its subsequent flux to carbohydrates accumulation in the mutant contributes to higher sugar and biomass productivity under enriched CO2 condition. The mutant was cultured outdoors in a photobioreactor with 6% CO2 aeration in hot summer season in southern Taiwan. The harvested biomass was subjected to separate hydrolysis and fermentation (SHF) for lactic acid production with carbohydrate concentration equivalent to 20 g/L glucose using the lactic acid-producing bacterium Lactobacillus plantarum 23. The conversion rate and yield of lactic acid were 80% and 0.43 g/g Chlorella biomass, respectively. CONCLUSIONS: These results demonstrated that the thermo-tolerant Chlorella mutant with high photosynthetic efficiency and biomass productivity under hot outdoor condition is an efficient fermentative feedstock for large-scale lactic acid production.

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%.

Nutrients and COD removal of swine wastewater with an isolated microalgal strain Neochloris aquatica CL-M1 accumulating high carbohydrate content used for biobutanol production.

In this study, a carbohydrate-rich microalga Neochloris aquatica CL-M1 was adapted to grow in swine wastewater. The effects of cultivation conditions (i.e., temperature, light intensity or N/P ratio) on COD/nutrients removal and carbohydrate-rich biomass production were investigated. The results indicate that the highest COD removal (81.7%) and NH3-N removal (96.2%) was achieved at 150µmolm-2s-1 light intensity, 25°C and N/P ratio=1.5/1. The highest biomass concentration and carbohydrate content was 6.10gL-1 and 50.46%, respectively, when N/P ratio=5/1. The resulting carbohydrate-rich microalgal biomass was pretreated and used as a feedstock for butanol fermentation. With the initial sugar concentration of 48.7gL-1 glucose and 3.4gL-1 xylose in the pretreated biomass, the butanol concentration, yield, and productivity were 12.0gL-1, 0.60molmol-1 sugar, and 0.89gL-1h-1, respectively, indicating the high potential of using Neochloris aquatica CL-M1 for butanol fermentation.

Perspectives on the feasibility of using microalgae for industrial wastewater treatment.

Although microalgae can serve as an appropriate alternative feedstock for biofuel production, the high microalgal cultivation cost has been a major obstacle for commercializing such attempts. One of the feasible solution for cost reduction is to couple microalgal biofuel production system with wastewater treatment, as microalgae are known to effectively eliminate a variety of nutrients/pollutants in wastewater, such as nitrogen/phosphate, organic carbons, VFAs, pharmaceutical compounds, textile dye compounds, and heavy metals. This review aims to critically discuss the feasibility of microalgae-based wastewater treatment, including the strategies for strain selection, the effect of wastewater types, photobioreactor design, economic feasibility assessment, and other key issues that influence the treatment performance. The potential of microalgae-bacteria consortium for treatment of industrial wastewaters is also discussed. This review provides useful information for developing an integrated wastewater treatment with microalgal biomass and biofuel production facilities and establishing efficient co-cultivation for microalgae and bacteria in such systems.

Enhancing bio-butanol production from biomass of Chlorella vulgaris JSC-6 with sequential alkali pretreatment and acid hydrolysis.

This study presents a successful butanol production method using alkali and acid pretreated biomass of Chlorella vulgaris JSC-6. The butanol concentration, yield, and productivity were 13.1g/L, 0.58mol/mol sugar, 0.66g/L/h, respectively. Nearly 2.93L/L of biohydrogen was produced during the acidogenesis phase in ABE fermentation. The hydrogen yield and productivity were 0.39mol/mol sugar and 104.2g/L/h respectively. In addition, the high glucose consumption efficiency (97.5%) suggests that the hydrolysate pretreated with NaOH (1%) followed by H2SO4 (3%) did not contain inhibitors to the fermentation. It was also discovered that an excess amount of nitrogen sources arising from hydrolysis of highly concentrated microalgal biomass negatively affected the butanol production. This work demonstrates the technical feasibility of producing butanol from sustainable third-generation feedstock (i.e., microalgal biomass).

Bio-butanol production from glycerol with Clostridium pasteurianum CH4: the effects of butyrate addition and in situ butanol removal via membrane distillation.

BACKGROUND: Clostridium pasteurianum CH4 was used to produce butanol from glycerol. The performance of butanol fermentation was improved by adding butyrate as the precursor to trigger the metabolic pathway toward butanol production, and by combining this with in situ butanol removal via vacuum membrane distillation (VMD) to avoid the product inhibition arising from a high butanol concentration. RESULTS: Adding 6 g L(-1) butyrate as precursor led to an increase in the butanol yield from 0.24 to 0.34 mol butanol (mol glycerol)(-1). Combining VMD and butyrate addition strategies could further enhance the maximum effective butanol concentration to 29.8 g L(-1), while the yield was also improved to 0.39 mol butanol (mol glycerol)(-1). The butanol concentration in the permeate of VMD was nearly five times higher than that in the feeding solution. CONCLUSIONS: The proposed butyrate addition and VMD in situ butanol removal strategies are very effective in enhancing both butanol titer and butanol yield. This would significantly enhance the economic feasibility of fermentative production of butanol. The VMD-based technology not only alleviates the inhibitory effect of butanol, but also markedly increases butanol concentration in the permeate after condensation, thereby making downstream processing easier and more cost-effective.

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.

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.

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.

Enhancing butanol production with Clostridium pasteurianum CH4 using sequential glucose-glycerol addition and simultaneous dual-substrate cultivation strategies.

Adding butyrate significantly enhanced butanol production from glycerol with Clostridium pasteurianum CH4, which predominantly produces butyrate (instead of butanol) when grown on glucose. Hence, the butyrate produced from assimilating glucose can be used to stimulate butanol production from glycerol under dual-substrate cultivation with glucose and glycerol. This proposed butanol production process was conducted by employing sequential or simultaneous addition of the two substrates. The latter approach exhibited better carbon source utilization and butanol production efficiencies. Under the optimal glucose to glycerol ratio (20 g L(-1) to 60 g L(-1)), the simultaneous dual-substrate strategy obtained maximum butanol titer, productivity and yield of 13.3 g L(-1), 0.28 g L(-1) h(-1), and 0.38 mol butanol/mol glycerol, respectively. Moreover, bagasse and crude glycerol as dual-substrates were also converted into butanol efficiently with a maximum butanol concentration, productivity and yield of 11.8 g L(-1), 0.14 g L(-1) h(-1), and 0.33 mol butanol/mol glycerol, respectively.

High yield bio-butanol production by solvent-producing bacterial microflora.

Highly efficient butanol-producing bacterial microflora were isolated from hydrogen-producing sludge of a sewage treatment plant. Based on denaturing gradient gel electrophoresis (DGGE) analysis and 16s rDNA comparison, four strains from the butanol-producing microflora were identified as Clostridium saccharoperbutylacetonicum, Clostridium butylicum, Clostridium beijernckii, and Clostridium acetobutylicum. The effects of glucose, FeSO(4) · 7H(2)O and yeast extract concentrations on the butanol production by the mixture culture were investigated on batch mode. The medium composition for bio-butanol production was optimized using the Box-Behnken design and response surface methodology (RSM). The maximum butanol production rate (0.25 ± 0.02 g/L-h) and concentration (12.4 g/L) were obtained under the condition of glucose concentration, 60 g/L; FeSO(4) · 7H(2)O, 0.516 g/L; yeast extract concentration, 5.13 g/L. Addition of 6.0 g/L butyric acid significantly increased the butanol titer to 17.51 ± 0.49 g/L. Pressurized fermentation strategy (employed with a 5L fermentor) further enhanced the butanol concentration to 21.35 g/L, along with a maximum butanol rate of 1.25 g/L-h.

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.

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.

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-ß-1,4-d-glucanase (80 and 100 kDa), exo-ß-1,4-d-glucanase (55 kDa) and ß-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-ß-1,4-d-glucanase, exo-ß-1,4-d-glucanase, ß-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 Clostridium pasteurianum 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.

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.