Microbial lipid production by the oleaginous yeast Cryptococcus curvatus O3 grown in fed-batch culture

This study investigates the capability of the oleaginous yeast Cryptococcus curvatus O3 to synthesize microbial lipids using glucose as its sole carbon source. Both glucose concentration and varying nitrogen sources have a significant effect on cell growth and microbial lipid accumulation in batch and fed-batch cultures. When cultivated in a shaking flask at 30 °C with glucose as sole carbon source, the cellular biomass and lipid content reached 51.8 kg m⁻³ and 651 g kg⁻¹, respectively. The fed-batch culture in a 30 × 10⁻³ m³ stirred-tank fermentor run for 185 h produced a cellular biomass, lipid content, and lipid productivity rate of up to 104.1 kg m⁻³, 827 g kg⁻¹, and 0.47 kg m⁻³ h⁻¹, respectively. These data indicate that C. curvatus O3 can be used as an ideal oleaginous yeast for microbial lipid production. Gas chromatography analysis of the synthesized microbial lipids revealed that the major constituents are long-chain fatty acids, such as palmitic acid, stearic acid, oleic acid, and linoleic acid. The results suggest that the microbial lipids produced by C. curvatus O3 can be used to produce biodiesel.     

Fermentative hydrogen production by Clostridium butyricum CGS5 using carbohydrate-rich microalgal biomass as feedstock

In this work, a carbohydrate-rich microalga, Chlorella vulgaris ESP6, was grown photoautotrophically to fix the CO₂. The resulting microalgal biomass was hydrolyzed by acid or alkaline/enzymatic treatment and was then used for biohydrogen production with Clostridium butyricum CGS5. The C. vulgaris biomass could be effectively hydrolyzed by acid pretreatment while similar hydrolysis efficiency was achieved by combination of alkaline pretreatment and enzymatic hydrolysis. The biomass of C. vulgaris ESP6 containing a carbohydrate content of 57% (dry weight basis) was efficiently hydrolyzed by acid treatment with 1.5% HCl, giving a reducing sugars (RS) yield of nearly 100%. C. butyricum CGS5 could utilize RS from C. vulgaris ESP6 biomass to produce hydrogen without any additional organic carbon sources. The optimal conditions for hydrogen production were 37 °C and a microalgal hydrolysate loading of 9 g RS/L with pH-controlled at 5.5. Under the optimal conditions, the cumulative H₂ production, H₂ production rate, and H₂ yield were 1476 ml/L, 246 ml/L/h, and 1.15 mol/mol RS, respectively. The results demonstrate that the C. vulgaris biomass has the potential to serve as effective feedstock for dark fermentative H₂ production.     

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

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

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.     

Direct hydrogen production from cellulosic waste materials with a single-step dark fermentation process

Biohydrogen production from cellulosic waste materials using dark fermentation is a promising technology for producing renewable energy. The purpose of this study was to evaluate residual cellulosic materials generated from local sources for their H₂ production potential without any pretreatment. Clostridium thermocellum ATCC 27405, a cellulolytic, thermophilic bacterium that has been shown to be capable of H₂ production on both cellobiose and α-cellulose substrates, was used in simultaneous batch fermentation experiments with dried distillers grain (DDGs), barley hulls (BH) and fusarium head blight contaminated barley hulls (CBH) as the carbon source. Overall, the dried distillers grain produced the highest concentration of hydrogen gas at 1.27 mmol H₂/glucose equivalent utilized. CBH and BH produced 1.18 and 1.24 mmol H₂/glucose equivalent utilized, respectively. Overall, this study indicates that hydrogen derived from a variety of cellulosic waste biomass sources is a possible candidate for the development of sustainable energy.     

Evaluation of the potential hydrogen production by diazotrophic Burkholderia species

Hydrogen (H₂) is considered one of the most promising fuels for sustainable energy. Because nitrogenase produces H₂ as a normal by-product, we tested the N₂-fixing bacterial strains Burkholderia unamae and Burkholderia tropica to determine their H₂ production capacities. To maximize H₂ production, several culture conditions were tested and optimized, including atmospheric conditions, carbon sources and chemical compounds such as enzyme cofactors and sugar cane molasses. The results showed that both strains were capable of H₂ production. The culture medium with the highest H₂ yield was composed of 1% v/v molasses enriched with Na₂MoO₄ (0.2 g/L), FeSO₄ (0.2 g/L) and cysteine (0.02 g/L) under a partial vacuum (air 20% v/v) without Ar final atmosphere. Under these conditions, the highest H₂ production rate obtained was 24.64 mmol H₂/L for B. unamae. The present study contributes an optimization process for H₂ production in N₂-fixing Burkholderia species. We propose further research and development to improve H₂ production rates in order to make biohydrogen a tangible reality.     

Photo-fermentative hydrogen gas production from dark fermentation effluent of acid hydrolyzed wheat starch with periodic feeding

Hydrogen gas production by photo-fermentation of dark fermentation effluent of acid hydrolyzed wheat starch was investigated at different hydraulic residence times (HRT = 1-10 days). Pure Rhodobacter sphaeroides (NRRL B-1727) culture was used in continuous photo-fermentation by periodic feeding and effluent removal. The highest daily hydrogen gas production (85 ml d⁻¹) was obtained at HRT = 4 days (96 h) while the highest hydrogen yield (1200 ml H₂ g⁻¹ TVFA) was realized at HRT = 196 h. Specific and volumetric hydrogen formation rates were also the highest at HRT = 96 h. Steady-state biomass concentrations and biomass yields increased with increasing HRT. TVFA loading rates of 0.32 g L⁻¹ d⁻¹ and 0.51 g L⁻¹ d⁻¹ resulted in the highest hydrogen yield and formation rate, respectively. Hydrogen gas yield obtained in this study compares favorably with the relevant literature reports probably due to operation by periodic feeding and effluent removal.     

Fermentative hydrogen production by Clostridium butyricum and Escherichia coli in pure and cocultures

The effect of coculture of Clostridium butyricum and Escherichia coli on hydrogen production was investigated. C. butyricum and E. coli were grown separately and together as batch cultures. Gas production, growth, volatile fatty acid production and glucose degradation were monitored. Whilst C. butyricum alone produced 2.09 mol-H₂/mol-glucose the coculture produced 1.65 mol-H₂/mol-glucose. However, the coculture utilized glucose more efficiently in the batch culture, i.e., it was able to produce more H₂ (5.85 mmol H₂) in the same cultivation setting than C. butyricum (4.62 mmol H₂), before the growth limiting pH was reached.     

Bacterial hydrogen production in recombinant Escherichia coli harboring a HupSL hydrogenase isolated from Rhodobacter sphaeroides under anaerobic dark culture

In this study, recombinant plasmid was constructed to analyze the effect of hydrogen production on the expression HupSL hydrogenase isolated from Rhodobacter sphaeroides in Escherichia coli. Although most of recombinant HupSL hydrogenase was produced as inclusion bodies the solubility of the protein increased significantly when the expression temperature shifted from 37 °C to 30 °C. Hydrogen production by expression of HupSL hydrogenase from recombinant E. coli increased 20.9-fold compared to control E. coli and 218-fold compared to wild type R. sphaeroides under anaerobic dark condition. The results demonstrate that HupSL hydrogenase, consisting of small and large subunits of hydrogenase isolated from R. sphaeroides, increases hydrogen production in recombinant E. coli. In addition conditions for enhancing the activity of HupSL hydrogenase in E. coli were suggested and were used to increase bacterial hydrogen production.     

High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana

Efficient conversion of glycerol waste from biodiesel manufacturing processes into biohydrogen by the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 was investigated. Biohydrogen production by T. neapolitana was examined using the batch cultivation mode in culture medium containing pure glycerol or glycerol waste as the sole substrate. Pre-treated glycerol waste showed higher hydrogen (H₂) production than untreated waste. Nitrogen (N₂) sparging and pH control were successfully implemented to maintain the culture pH and to reduce H₂ partial pressure in the headspace for optimal growth rate and to enhance hydrogen production from the glycerol waste. It was found that hydrogen production increased from 1.24 ± 0.06 to 1.98 ± 0.1 mol-H₂ mol⁻¹ glycerol_consumed by optimising N₂ sparging and pH control. We observed that in medium containing 0.05 M HEPES, with three cycles of N₂ sparging, the H₂ yield increased to 2.73 ± 0.14 mol-H₂ mol⁻¹ glycerol_consumed, which was 2.22-fold higher than the non-N₂ sparged H₂ yield (1.23 ± 0.06 mol-H₂ mol⁻¹ glycerol_consumed).     

Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation

Access to clean energy is vital to combat global warming and climate change, and nothing but hydrogen could better deliver it with ease to secure future energy needs. Biohydrogen could be produced in different routes including photolysis, water-gas shift reaction, dark, photo-fermentation and combination of both. Dark fermentative hydrogen production (DFHP) is efficient in comparison with photo-fermentation and utilizing organic waste ensures land usage and water for agriculture. Several microbes are involved in the process of biohydrogen production via dark fermentation and characterizing them at molecular level unveils holistic approach and understanding. Limited resources were available in terms of molecular tools for microbial characterization and this paper attempts to review the evolution of advanced molecular techniques including their merits and demerits. Understanding the composition of micro-flora is important in DFHP and could be classified as pure, co-cultures, enriched mixed cultures and mixed microbiota. These cultures act as seed sources for batch and continuous fermentations that help in understanding the efficiency of these methods. The schematics and systematic assessment of the various molecular tools (cloning, PCR-DGGE, FISH, NGS, CE-SSCP) for quantification, identification, detection and characterization of the microbial cell activity have been elaborated. Lastly, a comparative tabulation recapitulates the merits and drawbacks of each technique discussed. This provides valued information for choosing the right kind of microbial and molecular assessment tool for future characterization. Such analysis aids in suitable identification and characterization of microflora as potential biocatalysts for biohydrogen production through dark fermentation.     

Photofermentative production of hydrogen from organic acids by the purple sulfur bacterium Thiocapsa roseopersicina

A mutant strain of the anaerobic purple sulfur bacterium Thiocapsa roseopersicina, containing only nitrogenase as a functionally active enzyme for H₂ generation was utilized to study the production of H₂ from organic acids (acetate, pyruvate and succinate). Two types of potential substrates for H₂ production, thiosulfate and salts of various organic acids, were compared under photoheterotrophic growth conditions. Thiosulfate proved to be the preferred electron donor for T. roseopersicina; the consumption of organic acids became pronounced only following depletion of the thiosulfate supply. The system is suitable for the generation of H₂ from effluents of heterotrophic dark fermentation processes or waste streams rich in inorganic reduced sulfur compounds and/or simple organic acids.     

Neochloris oleoabundans grown on anaerobically digested dairy manure for concomitant nutrient removal and biodiesel feedstock production

Microalgae have been investigated as a promising biodiesel feedstock; however, large-scale production is not currently cost-competitive with petroleum diesel, and its environmental impacts have received little attention. Using wastewater to supply nutrients for algal growth obviates synthetic fertilizer use, provides on-site nutrient removal, and reduces greenhouse gas emissions. In this work, anaerobically digested dairy manure was used to grow the oleaginous green alga Neochloris oleoabundans. In batch culture experiments with both synthetic media and anaerobic digester effluent, N. oleoabundans assimilated 90-95% of the initial nitrate and ammonium after 6 d and yielded 10-30% fatty acid methyl esters on a dry weight basis. Cellular lipid content and the N concentration in the growth media were inversely correlated. In addition, the proportion of polyunsaturated fatty acids (i.e. C16:3, C18:2, and C18:3) decreased with N concentration over time while the proportion of C18:1 fatty acid increased. Although N deficiency is likely the primary driver behind lipid accumulation, the influence of culture pH confounded results and requires further study. Other living microorganisms in the digester effluent were not observed to affect algal growth and lipid productivity, though the breakdown of organic nitrogen may have hindered lipid accumulation traditionally achieved through the manipulation of synthetic media. This work highlights the potential for waste-grown mono-algal cultures to produce high quality biodiesel while accomplishing simultaneous wastewater treatment.     

Evaluation of hydrogen production by Rhodobacter sphaeroides O.U.001 and its hupSL deficient mutant using acetate and malate as carbon sources

Rhodobacter sphaeroides O.U.001 is one of the candidates for photobiological hydrogen production among purple non-sulfur bacteria. Hydrogen is produced by Mo-nitrogenase from organic acids such as malate or lactate. A hupSL in frame deletion mutant strain was constructed without using any antibiotic resistance gene. The hydrogen production potential of the R. sphaeroides O.U.001 and its newly constructed hupSL deleted mutant strain in acetate media was evaluated and compared with malate containing media. The hupSL⁻R. sphaeroides produced 2.42 l H₂/l culture and 0.25 l H₂/l culture in 15 mM malate and 30 mM acetate containing media, respectively, as compared to the wild type cells which evolved 1.97 l H₂/l culture and 0.21 l H₂/l culture in malate and acetate containing media, correspondingly. According to the results, hupSL⁻R. sphaeroides is a better hydrogen producer but acetate alone does not seem to be an efficient carbon source for photoheterotrophic H₂ production by R. sphaeroides.     

A potent lipid producing isolate of Epicoccum purpurascens AUMC5615 and its promising use for biodiesel production

Oils of oleaginous microorganisms are a powerful alternative to vegetable oils for biodiesel production. In this study, the fungus Epicoccum purpurascens AUMC5615 isolated from Egypt showed a potent high lipid content (80% lipid) when grown on 4% sucrose in submerged culture under continuous illumination. Under dark submerged conditions the lipid content has drastically decreased to 12%. In light static conditions, the lipid content was 70%; however, the net lipid yield was significantly lower than that of light submerged cultures because of the decrease in growth under light static conditions in comparison to light submerged cultures. Under dark static conditions the lipid content of the fungus has declined to 30%. These results indicate that light plays a crucial role in the lipid accumulation whereas submersion enhances the growth of the fungus. Concomitantly, the highest yield of carotenoids was obtained under light submerged conditions followed respectively by light static, dark submerged and dark static. This synchronized increase in carotenoids content might be implicated in protecting the high lipid pools in the fungus from peroxidation. Growing the fungus on 4% of crude molasses resulted in a net lipid production of 26.8 g per liter under light submerged conditions. The determination of fatty acids by GC/MS revealed that the major constituents are four saturated fatty acids, hexadecanoic, n-decanoic, dodecanoic and octadecanoic acids. These saturated fatty acids would give valuable stability properties of such fungal biodiesel. The current investigation opens the scope for the possible use of this promising fungal isolate in biodiesel production.     

Enhancement in hydrogen production by co-cultures of Bacillus and Enterobacter

Defined co-cultures of hydrogen (H₂) producers belonging to Citrobacter, Enterobacter, Klebsiella and Bacillus were used for enhancing the efficiency of biological H₂ production. Out of 11 co-cultures consisting of 2–4 strains, two co-cultures composed of Bacillus cereus EGU43, Enterobacter cloacae HPC123, and Klebsiella sp. HPC793 resulted in H₂ yield up to 3.0 mol mol⁻¹ of glucose. Up-scaling of the reactor by 16-fold resulted in a corresponding increase in H₂ production with an actual evolution of 7.44 L of H₂. It constituted 58.2% of the total biogas. Continuous culture evolution of H₂ by co-cultures (B. cereus EGU43 and E. cloacae HPC123) immobilized on ligno-cellulosic materials resulted in 6.4-fold improvement in H₂ yield compared to free floating bacteria. This synergistic influence of B. cereus and E. cloacae can offer a better strategy for H₂ production than undefined or mixed cultures.     

Dark fermentative hydrogen production with crude glycerol from biodiesel industry using indigenous hydrogen-producing bacteria

Glycerol is an inevitable by-product from biodiesel synthesis process and could be a promising feedstock for fermentative hydrogen production. In this study, the feasibility of using crude glycerol from biodiesel industry for biohydrogen production was evaluated using seven isolated hydrogen-producing bacterial strains (Clostridium butyricum, Clostridium pasteurianum, and Klebsiella sp.). Among the strains examined, C. pasteurianum CH4 exhibited the best biohydrogen-producing performance under the optimal conditions of: temperature, 35 °C; initial pH, 7.0; agitation rate, 200 rpm; glycerol concentration, 10 g/l. When using pure glycerol as carbon source for continuous hydrogen fermentation, the average H₂ production rate and H₂ yield were 103.1 ± 8.1 ml/h/l and 0.50 ± 0.02 mol H₂/mol glycerol, respectively. In contrast, when using crude glycerol as the carbon source, the H₂ production rate and H₂ yield was improved to 166.0 ± 8.7 ml/h/l and 0.77 ± 0.05 mol H₂/mol glycerol, respectively. This work demonstrated the high potential of using biodiesel by-product, glycerol, for cost-effective biohydrogen production.     

Bio-hydrogen production by photo-fermentation of dark fermentation effluent with intermittent feeding and effluent removal

Pure culture of Rhodobacter sphaeroides (NRRL- B1727) was used for continuous photo-fermentation of volatile fatty acids (VFA) present in the dark fermentation effluent of ground wheat starch. The feed contained 1950 ± 50 mg L⁻¹ total VFA with some nutrient supplementation. Hydraulic residence time (HRT) was varied between 24 and 120 hours. The highest steady-state daily hydrogen production (55 ml d⁻¹) and hydrogen yield (185 ml H₂ g⁻¹ VFA) were obtained at HRT = 72 hours (3 days). Biomass concentration increased with increasing HRT. Volumetric and specific hydrogen formation rates were also maximum at HRT = 72 h. High extent of TVFA fermentation at HRT = 72 h resulted in high hydrogen gas production.