Oleaginous yeast Cryptococcus curvatus culture with dark fermentation hydrogen production effluent as feedstock for microbial lipid production

Volatile fatty acids (VFA) from dark fermentation hydrogen production were tested as carbon sources for the culture of oleaginous yeast Cryptococcus curvatus, which is a promising feedstock for biofuel production. The optimal acetate concentration and pH were investigated when potassium acetate was used as the sole carbon source. Comparisons were then made when hydrogen production effluent (HPE) from synthetic wastewater was tested as feedstock. A pH-stat culture fed with acetic acid ultimately produced 168 g/L biomass, with a lipid content of 75.0%. No inhibitor to yeast growth was produced in the hydrogen production process. However, inhibition occurred in culture with HPE from food waste (FW), indicating that inhibitors may be present in the original raw food waste. This inhibition could be avoided by a process that uses glucose as the initial carbon source and then is continuously fed with FW-HPE. The biomass productivity in this continuous culture process reached 0.34 g/L/h, but the lipid content was only 13.5%. These results suggest that FW-HPE alone is not an optimal feedstock, but HPE derived from nitrogen-deficient waste streams could be good feedstocks. This study provides preliminary evidence for the feasibility of using organic waste for the co-production of hydrogen and lipid.     

Genetic variability in Cynara cardunculus L. domestic and wild types for grain oil production and fatty acids composition

This paper aimed to study the genetic variability within different types of Cynara cardunculus L., domestic and wild types, for their grain oil amount and oil fatty acid composition.The grain oils were extracted from 8 domestic cardoons and 4 wild cardoons, by Soxhlet method, and obtained oils were characterized for palmitic, stearic, oleic and linoleic acids by gas chromatography.The oil amount, resulted on average of accessions 216 g kg⁻¹ DM with a good range of variability (CV = 11.7%). Unsaturated acids (oleic and linoleic) predominated over saturated ones (stearic and palmitic acids), the chemical characterization of extracted oil, showed the main compound (as % of analysed fatty acids), averaged for all populations, was linoleic acid (44.5%), followed by oleic acid (42.6%), palmitic acid (9.8%) and stearic acid (3.1%). In particular referring the oleic acid wild cardoon populations showed a mean value of 289 g kg⁻¹ oil, against a mean value of 472 g kg⁻¹ oil showed by domestic cardoon accessions. Three of the studied domestic cardoon (‘DC1’, ‘DC3’ and ‘DC7’) showed values higher than 795 g kg⁻¹ oil, while all the other accessions had concentration lower than 370 g kg⁻¹ oil.The three types of domestic cardoon ‘DC1’, ‘DC3’ and ‘DC7’ showed a fatty acids profile similar to genetic modified sunflower oil, representing new genetic material that potentially could be used for high quality biodiesel production, characterised by a low Iodine Number.     

Enhancement of phototrophic hydrogen production by Rhodobacter sphaeroides ZX-5 using fed-batch operation based on ORP level

In this study, a new outer-cycle flat-panel photobioreactor was designed for an anaerobic, photo-fermentation process by Rhodobacter sphaeroides ZX-5. In order to obtain the high hydrogen yield, photo-hydrogen production by fed-batch culture with on-line oxidation-reduction potential (ORP) feedback control was investigated. Meanwhile, the effects of feeding malic acid concentration and pH adjustment on the growth and hydrogen production of R. sphaeroides ZX-5 were studied. In the entire fed-batch culture, biomass (i.e., OD₆₆₀) rapidly increased up to 1.79 within 18 h, and then OD₆₆₀ value stayed constant within a range of 1.85-2.18 until the end of the photo-fermentation. The cumulative hydrogen volumes in each phase of fed-batch process were 2339, 1439, 1328, and 510 ml H₂/l-culture, respectively. Throughout the entire repeated fed-batch photo-fermentation, the maximum substrate conversion efficiency of 73.03% was observed in the first fed-batch process, obviously higher than that obtained from batch culture process (59.81%). In addition, compared to the batch culture, a much higher maximum hydrogen production rate (102.33 ml H₂/l h) was achieved during fed-batch culture. The results demonstrated that photo-hydrogen production using fed-batch operation based on ORP feedback control is a favorable choice of sustainable and feasible strategy to improve phototrophic hydrogen production efficiency.     

Thermophilic hydrogen fermentation using Thermotoga neapolitana DSM 4359 by fed-batch culture

Biohydrogen fermentation by the hyperthermophile Thermotoga neapolitana was conducted in a continuously stirred anaerobic bioreactor (CSABR). The production level of H₂ from fermentation in a batch culture with pH control was much higher than without pH control from pentose (xylose) and hexose (glucose and sucrose) substrates. The respective H₂ yield in the batch culture with pH control from xylose and glucose was 2.22 ± 0.11 mol-H₂ mol⁻¹ xylose_consumed and 3.2 ± 0.16 mol-H₂ mol⁻¹ glucose_consumed, which was nearly 1.2-fold greater for xylose and 1.6-fold greater for glucose than without pH control. In the case of sucrose, the H₂ yield from fermentation increased by 40.63%, compared with fermentation in batch cultures without pH control, from 3.52 ± 0.171 to 4.95 ± 0.25 mol-H₂ mol⁻¹ sucrose_consumed. The effects of stirring speed and different pH levels on growth and H₂ production were studied in the CSABR for highly efficient H₂ production. Growth and H₂ production of this bacterial strain in a batch culture with pH control or without pH control using a 3 L bioreactor was limited within 24 h due to substrate exhaustion and a decrease in the culture’s pH. The pH-controlled fed-batch culture with a xylose substrate added in doses was studied for the prevention of substrate-associated growth inhibition by controlling the nutrient supply. The highest H₂ production rates were approximately 4.6, 4.1, 3.9, and 4.3 mmol-H₂ L⁻¹ h⁻¹ at 32, 52, 67, and 86 h, respectively.     

Production of microbial oil with high oleic acid content by Trichosporon capitatum

Microbial oils with high unsaturated fatty acids content, especially oleic acid content, are good feedstock for high quality biodiesel production. Trichosporon capitatum was found to accumulate lipid with around 80% oleic acid and 89% total unsaturated fatty acids content on nitrogen-limited medium. In order to improve its lipid yield, effects of medium components and culture conditions on cell growth and lipid accumulation were investigated. Optimization of media resulted in a 61% increase in the lipid yield of T. capitatum after cultivation at 28 °C and 160 rpm for 6 days. In addition, T. capitatum could grow well on cane molasses and afford a lipid yield comparable to that on synthetic nitrogen-limited medium. The biodiesel from the microbial oil produced by T. capitatum on cane molasses displayed a low cold filter plugging point (−15 °C), and so T. capitatum might be a promising strain to provide lipid suitable for high quality biodiesel production.     

Effects of furfural and acetic acid on growth and lipid production from glucose and xylose by Rhodotorula glutinis

Microbial conversion of lignocellulosic sugars to triacylglycerols (a biodiesel or renewable diesel feedstock) was investigated using the oleaginous yeast Rhodotorula glutinis (ATCC 15125). In the shake flask experiments, R. glutinis was first grown in a nitrogen-rich medium utilizing an artificial acid hydrolysate of lignocellulosic biomass switchgrass as the sole carbon and energy source. Once the culture had reached the stationary phase, the cells were harvested and transferred to a fresh nitrogen-free media containing artificial acid hydrolysate sugars for lipid accumulation. Analysis of the data collected showed that the yeast were able to grow in the medium containing artificial acid hydrolysate sugars as the carbon and energy source. The net specific Growth rate(s) indicated that the presence of acetic acid and furfural in the artificial acid hydrolysate inhibited the growth of R. glutinis on glucose, but not the growth on xylose. The lipid accumulated in the cells, determined by gravimetrical method, increased from initial 4.3%-39.0% of dry cell mass weight. The major fatty acids of the accumulated lipids were palmitic acid, stearic acid, oleic acid, linoleic acid and ??-linoleic acid. These results indicate that it is feasible to convert the sugars in acid hydrolysate of lignocellulosic biomass to triacylglycerols using R. glutinis.     

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.     

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.     

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.     

Net energy analysis of the production of biodiesel and biogas from the microalgae: Haematococcus pluvialis and Nannochloropsis

Microalgae have been proposed as possible alternative feedstocks for the production of biodiesel because of their high photosynthetic efficiency. The high energy input required for microalgal culture and oil extraction may negate this advantage, however. There is a need to determine whether microalgal biodiesel can deliver more energy than is required to produce it. In this work, net energy analysis was done on systems to produce biodiesel and biogas from two microalgae: Haematococcus pluvialis and Nannochloropsis. Even with very optimistic assumptions regarding the performance of processing units, the results show a large energy deficit for both systems, due mainly to the energy required to culture and dry the microalgae or to disrupt the cell. Some energy savings may be realized from eliminating the fertilizer by the use of wastewater or, in the case of H. pluvialis, recycling some of the algal biomass to eliminate the need for a photobioreactor, but these are insufficient to completely eliminate the deficit. Recommendations are made to develop wet extraction and transesterification technology to make microalgal biodiesel systems viable from an energy standpoint.     

Bio-hydrogen production enhancement by co-cultivating Rhodopseudomonas palustris WP3-5 and Anabaena sp. CH3

Photobiological H₂ production is a promising method for renewable energy development. An innovative system that co-cultivating Rhodopseudomonas palustris WP3-5 and Anabaena sp. CH3 was carried out to estimate the effect of co-cultivation on H₂ production enhancement. H₂ production prolongation and enhancement were observed due to the light and metabolic compatibility of these two strains. Co-culture system served by acetate and fructose as carbon source can accumulate H₂ in 140.8 mL, almost double than the sum of individuals. Moreover, the enhancement of H₂ production was significantly affected by the mixed ratio of two strains. The mixed ratio (WP3-5:CH3) of 1:2 showed a highest H₂ production rate in 44.8 mL-H₂/L-culture/h, and both 2:1 and 1:2 exhibited a relatively high substrate conversion efficiency during the latest period of cultivation, whereas the mixed ratio of 1:1 and 3:1 only revealed a prolongation in H₂ production due to metabolic compatibility of two strains.     

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).     

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

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

Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3

A hydrogen producing strain newly isolated from anaerobic sludge in an anaerobic bioreactor, was identified as Clostridium beijerinckii Fanp3 by 16S rDNA gene sequence analysis and detection by BioMerieux Vitek. The strain could utilize various carbon and nitrogen sources to produce hydrogen, which indicates that it has the potential of converting renewable wastes into hydrogen. In batch cultivations, the optimal initial pH of the culture medium was between 6.47 and 6.98. Using 0.15 M phosphate as buffer could alleviate the medium acidification and improve the overall performance of C. beijerinckii Fanp3 in hydrogen production. Culture temperature of 35 °C was established to be the most favorable for maximum rate of hydrogen production. The distribution of soluble metabolic products (SMP) was also greatly affected by temperature. Considering glucose as a substrate, the activation energy (E_a) for hydrogen production was calculated as 81.01 kcal/mol and 21.4% of substrate energy was recovered in the form of hydrogen. The maximal hydrogen yield and the hydrogen production rate were obtained as 2.52 mol/mol-glucose and 39.0 ml/g-glucose h⁻¹, respectively. These results indicate that C. beijerinckii Fanp3 is an ideal candidate for the fermentative hydrogen production.     

Hydrogen gas production by combined systems of Rhodobacter sphaeroides O.U.001 and Halobacterium salinarum in a photobioreactor

Rhodobacter sphaeroides O.U.001 is a photosynthetic non-sulfur bacterium which produces hydrogen from organic compounds under anaerobic conditions. Halobacterium salinarum is an archaeon and lives under extremely halophilic conditions (4 M NaCl). H. salinarum contains a retinal protein bacteriorhodopsin in its purple membrane which acts as a light-driven proton pump. In this study the Rhodobacter sphaeroides O.U.001 culture was combined with different amounts of packed cells of H. salinarum S9 or isolated purple membrane fragments in order to increase the photofermentative hydrogen gas production. The packed cells of H. salinarum have the ability to pump protons upon illumination due to the presence of bacteriorhodopsin. The proton gradient produced may be used for the formation of ATP or protons may be used for H₂ production by R. sphaeroides. Similar to intact cells purple membrane fragments may also form vesicles around certain ions and may act like closed systems.     

Chlorella sp.: A new strain with highly saturated fatty acids for biodiesel production in bubble-column photobioreactor

The biodiesel production from a naturally isolated strain of Chlorella in 2 L bubble-column photobioreactor was studied. The microalgal strain was isolated from the rice paddy-field soil samples during a screening program. After 17 days, at the end of exponential phase of growth, the total content of the lipids was extracted. The extracted fatty acids were first esterified and then identified using GC/MS analysis. Several types of fatty acid methyl esters (FAMEs) were identified in the isolated microalga and the presence of saturated fatty acids in Chlorella sp. MCCS 040 was approved. The composition of fatty acids in the studied species of microalga was mainly palmitic acid methyl ester, myristic acid methyl ester, stearic acid methyl ester and undecanoic acid methyl ester. This strain because of its highly saturated fatty acids content can be an ideal candidate for biodiesel production.     

The influence of seed and oil storage on the acid levels of rubber seed oil, derived from Hevea brasiliensis grown in Xishuangbanna, China

High acid levels, characteristic of rubber seed oil (RSO), limit RSO use in biodiesel production. The aims of this study were to determine the causes of these high acid levels by investigating what affects the storage of rubber seeds and RSO had on the acid levels. Two storage conditions/methods were evaluated, one representing a proposed storage method (SM 1), the other mimicking storage conditions characteristic to the Xishuangbanna region (SM 2). Furthermore, RSO storage was evaluated by testing RSO acid levels over a 2-month period, under standard storage conditions. Seeds from SM 2 displayed increased seed pile temperatures, higher levels of Mildew infection, lower seed oil content and higher acid levels. Low seed oil content and high acid values of SM 2 were resultant of the high Mildew infection and increased seed pile temperatures. In addition, a critical value of 90% relative humidity of seed piles was identified, above which Mildew infection increased sharply. Storage of crude RSO resulted in increased acid values. This data shows that in order to reduce high acid values, seed pile temperature, humidity and Mildew infections need to be kept to a minimum, as well as the storage time of the seeds and the RSO.     

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.