Oil assessment of Halamphora coffeaeformis diatom growing in a hybrid two-stage system for biodiesel production

The lipid content, composition and productivity of the marine benthic diatom Halamphora coffeaeformis were studied in order to evaluate its potential as feedstock for biodiesel production. Cultures were carried out in two stages: I) in photobioreactors (PBRs) to increase the biomass as inoculum for larger volumes, and II) in raceway ponds to increase naturally the triacylglycerol (TAG) content during the stationary growth phase. Biomass concentrations of 0.64 g L⁻¹ and 0.23 g L⁻¹ were reached in the PBR and the raceway pond, respectively. Total lipid content was 54.4 (±11.6) % ash free dry weight (AFDW) in the raceway pond on day 19 (harvest day), with a neutral lipid content of 34% AFDW. The TAG productivity in the raceway pond was 1.2 mg L⁻¹ d⁻¹. The indicators of biodiesel, calculated from fatty acid profile composition, showed that H. coffeaeformis oil was of good quality, according to international standards. Some hypothetical aspects are proposed in order to improve lipid productivity and net energy ratio in processes at larger scales.     

Enhancement of biomass production in Scenedesmus bijuga high-density culture using weakly absorbed green light

To increase microalgae biomass production and support high density cultures in photobioreactors artificial illumination systems have been designed to increase photosynthetic activity. Supplemental lighting systems are commonly composed by a combination of chlorophyll (a + b) strongly absorbed wavelengths, while weakly absorbed wavelengths are not present. At this work we compared the photosynthetic activity and biomass production induced by chlorophyll (a + b) strongly versus weakly absorbed wavelengths in Scenedesmus bijuga microalgae cultures at different biomass densities. Photosynthetic activity and biomass production induced by 4 different wavelengths using LEDs (blue – peak at λ470 nm; green – peak at λ530 nm; red – peak at λ655 nm; and white-4100 K) were measured and analyzed on high-density cultures of S. bijuga. As culture density increased the chlorophyll (a + b) weakly absorbed green light penetrated deeper into the samples inducing higher oxygen evolution at culture concentration of 1.45 g L⁻¹ compared to the chlorophyll (a + b) strongly absorbed red light. High-density culture (2.19 g L⁻¹) cultivated under green light showed higher biomass production rate (30 mg L⁻¹ d⁻¹) with a 8.43% biomass growth in a 6-day period compared to the same quantum flux of red light that induced 4.35% biomass growth on the same period. The integration of green LEDs into photobioreactors lighting apparatus could improve the existing systems composed predominantly by red and blue LEDs increasing biomass productivity of high-density cultures at latter stages of microalgae cultivation.     

Assessment of triacylglycerol content in Chlorella vulgaris cultivated in a two-stage process

Chlorella vulgaris cultivation in two-stage process was applied to increase the lipid productivity without compromising the biomass productivity. At the first stage, microalgae was cultivated under nutrient sufficient conditions to obtain a maximized cell density; at the second stage, nitrate conditions are changed to trigger the accumulation of TAG. During first stage, the maximum biomass productivity (32 mg L⁻¹ d⁻¹) was observed after 13 days under nutrient sufficient conditions with 1.21 g L⁻¹ NaNO₃ and 0.00449 g L⁻¹ K₂HPO₄. Maximum lipid content (25.4% DW), lipid productivity (7.5 mg L⁻¹ d⁻¹) and TAG content (41.3% in total lipids) were favored by the nitrogen starvation conditions for more 4 days, at the second stage. Oil extracted at the second stage contained lower percentage of PUFAs being more suitable for the biodiesel production when compared with the oil extracted at the first stage. This two-stage phototrophic process is promising to provide a more efficient way for on a large-scale production of algal biomass and biodiesel production.     

Lignocellulosic ethanol production employing immobilized Saccharomyces cerevisiae in packed bed reactor

Most of ethanol production processes are limited by lower ethanol production rate and recyclability problem of ethanologenic organism. In the present study, immobilized co-fermenting Saccharomyces cerevisiae GSE1618 was employed for ethanol fermentation using rice straw enzymatic hydrolysate in a packed bed reactor (PBR). The immobilization of S. cerevisiae was performed by entrapment in Ca-alginate for optimization of ethanol production by varying alginic acid concentration, bead size, glucose concentration, temperature and hardening time. Remarkably, extra hardened beads (EHB) immobilized with S. cerevisiae could be used up to repeated 40 fermentation batches. In continuous PBR, maximum 81.82 g L⁻¹ ethanol was obtained with 29.95 g L⁻¹ h⁻¹ productivity with initial glucose concentration of 180 g L⁻¹ in feed at dilution rate of 0.37 h⁻¹. However, maximum ethanol concentration of 40.33 g L⁻¹ (99% yield) with 24.61 g L⁻¹ h⁻¹ productivity was attained at 0.61 h⁻¹ dilution rate in fermentation of un-detoxified rice straw enzymatic hydrolysate (REH). At commercial scale, EHB has great potential for continuous ethanol production with high productivity using lignocellulosic hydrolysate in PBR.     

A sequential process of anaerobic solid-state fermentation followed by dark fermentation for bio-hydrogen production from Chlorella sp.

Microalgal biomass has recently been one of the most widely studied feedstocks for bio-hydrogen production, owing to its richness in fermentable components, e.g. polysaccharides and proteins, and high biomass productivity. In this study, biomass of microalga Chlorella sp. TISTR 8411 was converted to hydrogen through a sequential process consisting of an anaerobic solid-state fermentation (ASSF) followed by a dark fermentation. The microalga was grown photoautothrophically in 80-L rectangular glass tanks and then scaled-up to a 240-L open pond for the production of biomass. The highest biomass concentration attained was 4.45 g L⁻¹. The biomass was harvested with over 90% flocculation efficiency at pH 11.5 and a biomass concentration of 2.6 g/L. The sequential process gave a total hydrogen yield (HY) of 16.2 mL/g-volatile-solid (VS), of which 11.6 mL/g-VS was from ASSF. The high HY obtained from the ASSF indicated that it was effective and could be integrated with a conventional hydrogen production process to improve energy recovery from biomass.     

Effect of carbon supply mode on biomass and lipid in CSMCRI's Chlorella variabilis (ATCC 12198)

CSIR-CSMCRI's Chlorella variabilis (ATCC 12198) was evaluated through autotrophic, mixotrophic and heterotrophic growth for lipid production. Autotrophic growth was assessed by providing sodium bicarbonate/sodium carbonate/CO₂ (air in a medium). Higher lipid productivity (115.94 mg L⁻¹ d⁻¹) with higher biomass productivity (724.98 mg L⁻¹ d⁻¹) of this strain was attained through bicarbonate and CO₂ sequestration in a photobioreactor. Ability to regulate the pH in favorable bicarbonate/carbonate ratio showed its potential in alkaline effluent based carbon sequestration system for biofuel generation. The simultaneous study was also conducted to understand the effect of elevated CO₂ (0.4, 1 and 1.2 g L⁻¹) in air on the culture to assess adaptation, growth and lipid in the closed chamber conditions. It was observed that CO₂ sequestration by the microalgae from the CO₂ enriched environment was optimum at 1 g L⁻¹ C. variabilis adapted to comparatively higher CO₂ (1 g L⁻¹) but grew better in low CO₂ (0.4 g L⁻¹). It was also observed that the growth, lipid content and fatty acid composition was significantly affected by CO₂ supply strategies. The effect of intermittently added sodium bicarbonate at different pH on microalgal lipid content and composition of fatty acids was observed which could affect the quality of biodiesel. The effect on fatty acid composition was observed in response to carbon supply mode during the microalgal growth at different pH dictating the properties of biodiesel.     

Ethanol production from halophyte Juncus maritimus using freezing and thawing biomass pretreatment

Juncus maritimus contains (41.5 ± 0.3)% cellulose and (31.34 ± 0.2)% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for ethanol production. Dilute acid or freezing/thawing pretreatments and enzymatic saccharification were evaluated for conversion of halophyte plant from J. maritimus cellulose and hemicelluloses to monomeric sugars. The maximum concentration of released glucose from J. maritimus (53.78 ± 3.24) g L⁻¹) by Freezing/thawing pretreatment and enzymatic saccharification (55 °C, pH 5.0 and 48 h) using CellicCTec2 from Novozymes and (49.14 ± 5.24) g L⁻¹ obtained by dilute acid pretreatment. The maximum yield of ethanol from acid pretreated enzyme saccharified J. maritimus hydrolyzate by Saccharomyces cerevisiae strain was (84.28 ± 5.11)% of the theoretical yield with a productivity of (0.88 ± 0.16)g L⁻¹ h⁻¹. It was (90.87 ± 1.94)% of the theoretical yield with a productivity of (1.04 ± 0.10) g L⁻¹h⁻¹ for freezing/thawing pretreated plant and enzymatic hydrolysis by CellicCTec2.     

Efficient ethanol production from inulin by two-stage aerate strategy

A major concern for ethanol production from inulin-containing materials, is the higher unconverted sugar, which increases the cost of ethanol production and wastewater treatment. Some key factors, such as inulinase, biomass or aeration rates, were studied to solve the problems in the process of ethanol fermentation from inulin. It was showed that more inulinase and increasing inoculum size can shorten the fermentation time, but could not reduce residual sugars. Two-stage aerate strategy was developed to utilize the remained sugars: keep the aeration at 5 h⁻¹ at the first 12 h, and drop it to 1.2 h⁻¹. Under this condition, contradiction between fermentation time and high ethanol yield was solved (60 h and 0.43 g g⁻¹), and the final residual sugar concentration decreased to about 10 g L⁻¹ with 98 g L⁻¹ ethanol. The ethanol productivity was up to 1.63 g L⁻¹ h⁻¹, which is the highest productivity of ethanol fermentations from inulin-containing materials.     

Effect of salt and sodium concentration on the anaerobic methanisation of the halophyte Tripolium pannonicum

The halophyte species Sea Aster (Tripolium pannonicum) was grown with different concentrations of artificial seawater. In a second experiment, T. pannonicum was cultivated with a nutrient solution containing different concentrations of NaCl. This halophyte biomass was used to determine the biogas production potential. According to the findings, it is possible to produce high yields of methane using biomass from halophytes cultivated in the presence of salt. Biogas and methane yield are influenced by the salt content of the plant tissue, however, high concentrations of salt in the anaerobic reactors itself inhibit the biogas and methane production. The highest methane yield is obtained using plant substrates grown at 22.5 g L⁻¹ sea-salt with a value of 313 cm³ g⁻¹ of VS. When treating T. pannonicum with different concentrations of NaCl, biogas and methane yields are highest when using plant substrates grown at 30 g L⁻¹ to produce values of 554 cm³ g⁻¹ of VS and 447 cm³ g⁻¹ of VS, respectively. Other research was carried out to study the effect of sodium on the biogas and methane yields using substrate from T. pannonicum cultured under non-saline conditions and adding different amounts of NaCl to the anaerobic reactors. Adding NaCl to the reactors decreases the biogas and methane production but using a salt-adapted inoculum increases the biogas yield in comparison to the non-adapted inoculum.     

Effectiveness of dilute oxalic acid pretreatment of Miscanthus × giganteus biomass for ethanol production

In the present study the effect of temperature, reaction time and dilute oxalic acid (OA) concentration during steam-pretreatment of Miscanthus × gigantueus has been evaluated using the combined severity factor (CS). At the highest CS glucan and lignin content in the water insoluble fraction (WIF) increased, while xylan content decreased. While glucose recovery in the water soluble fraction (WSF) was found at low concentration when mild CS were used (≤5.0 g L⁻¹ at CS ≤ 2.17), xylose and arabinose concentrations were higher at low-mild CS (1.58–2.17) with a concentration peak at CS 2.03 (39.9 and 3.2 g L⁻¹ for xylose and arabinose, respectively). The decrease in pentoses coincided with inhibitory formation in the WSF, namely acetic acid, furfural, HMF and phenolic compounds. Glucan conversion rose from 46.1% at CS 1.54 to 91.2% at CS 2.76. Likewise, maximum ethanol concentration was achieved at CS 2.76, corresponding to 20.2 g L⁻¹ and a volumetric ethanol productivity of 0.28 g L⁻¹ h⁻¹. Negative correlations have been found between xylan vs. glucan conversion and xylan vs. ethanol production, suggesting that decreasing the xylan content in WIF increases both saccharification rate and ethanol concentration (R² 0.91 and R² 0.93, respectively). On the other hand, a positive correlation was found between ethanol production and glucan conversion (R² 0.93). Fermentation of WSF by Scheffersomyces (Pichia) stipitis CBS 6054 at CS 1.54 produced 12.1 g L⁻¹ of ethanol after 96 h incubation with a volumetric ethanol productivity of 0.13 g L⁻¹ h⁻¹.     

Production of ethanol from raw juice and thick juice of sugar beet by continuous ethanol fermentation with flocculating yeast strain KF-7

Sugar beet juice can serve as feedstock for ethanol product due to its high content of fermentable sugars and high energy output/input ratio. Batch ethanol fermentation of raw juice and thick juice proved that addition of mineral nutrients could not improve ethanol concentration, but could accelerate the fermentation rate. Fermentation of thick juice with an initial pH of 9.1 did not affect the fermentation process. The continuous ethanol fermentation of raw juice was performed at 35 °C with a dilution rate of 0.3 h⁻¹, resulting in ethanol concentration, ethanol yield and productivity of 70.7 g L⁻¹, 89.8% and 21.2 g L⁻¹ h⁻¹, respectively. A two-stage reactor was used in the continuous ethanol fermentation of thick juice by feeding fresh yeast cells into the second reactor. This process was stable at a total process dilution rate of 0.11 h⁻¹ with an overall sugar concentration of 190 g L⁻¹ in the influent. The ethanol concentration was kept at approximately 80 g L⁻¹, corresponding to ethanol yield of 82.5% and productivity of 8.8 g L⁻¹ h⁻¹.     

Hydrogen production by Escherichia coli during anaerobic utilization of mixture of lactose and glycerol: Enhanced rate and yield,prolonged production

Escherichia coli wild type has the ability to utilize lactose or the mixture of lactose and glycerol producing bio-hydrogen (H₂) at different pH values. At pH 7.5 in hyaB (lacking large subunit of hydrogenase (Hyd)-1) and hybC (lacking large subunit of Hyd-2) single mutants fermenting lactose (1 g L⁻¹) H₂ yield was ∼7- and 5-fold more, respectively, compared to the wild type. During the fermentation of lactose (1 g L⁻¹) and glycerol (10 g L⁻¹) mixture H₂ yield in wild type increased ∼3-fold, compared to fermenting lactose only. H₂ generation in wild type was monitored in batch cultures during 168 h of growth when utilizing the mixture of lactose and glycerol in all combinations of different concentration. In hyaB but not in hybC mutant H₂ evolution was detected till 240 h in the mixture of 5 g L⁻¹ lactose and 10 g L⁻¹ glycerol. The highest H₂ production rate of 21.94 mL L⁻¹ h⁻¹ was detected in hyaB mutant at pH 7.5 when 1 g L⁻¹ lactose was applied. The results showed optimized H₂ production using different mutants, lactose and its mixture with glycerol. They can be applied for renewable energy, especially bio-H₂ production.     

Production of butanol (a biofuel) from agricultural residues: Part II – Use of corn stover and switchgrass hydrolysates

Acetone butanol ethanol (ABE) was produced from hydrolysed corn stover and switchgrass using Clostridium beijerinckii P260. A control experiment using glucose resulted in the production of 21.06 g L^?1 total ABE. In this experiment an ABE yield and productivity of 0.41 and 0.31 g L^?1 h^?1 was achieved, respectively. Fermentation of untreated corn stover hydrolysate (CSH) exhibited no growth and no ABE production; however, upon dilution with water (two fold) and wheat straw hydrolysate (WSH, ratio 1:1), 16.00 and 18.04 g L^?1 ABE was produced, respectively. These experiments resulted in ABE productivity of 0.17–0.21 g L^?1 h^?1. Inhibitors present in CSH were removed by treating the hydrolysate with Ca(OH)₂ (overliming). The culture was able to produce 26.27 g L^?1 ABE after inhibitor removal. Untreated switchgrass hydrolysate (SGH) was poorly fermented and the culture did not produce more than 1.48 g L^?1 ABE which was improved to 14.61 g L^?1. It is suggested that biomass pretreatment methods that do not generate inhibitors be investigated. Alternately, cultures resistant to inhibitors and able to produce butanol at high concentrations may be another approach to improve the current process.     

ABE fermentation from enzymatic hydrolysate of NaOH-pretreated corncobs

Acetone butanol ethanol (ABE) was produced from enzymatic-hydrolyzed corncobs by Clostridium saccharobutylicum DSM 13864. Pretreatment of corncobs was carried out with 0.5 mol L⁻¹ NaOH followed by enzymatic hydrolysis. The yield of total reducing sugars was 917 g kg⁻¹ pretreated (de-lignified) and washed corncobs. The hydrolysate was used without sediments removal for ABE fermentation. A solvent production of 19.44 g L⁻¹ with 12.27 g L⁻¹ butanol was obtained from 55.22 g L⁻¹ sugars, resulting in an ABE yield of 350 g kg⁻¹ and a production rate of 0.54 g L⁻¹ h⁻¹. A control experiment using 55.3 g L⁻¹ mixed sugars resulted in an ABE production of 16.81 g L⁻¹ with 10.26 g L⁻¹ butanol, corresponding to an ABE yield of 300 g kg⁻¹ and a production rate of 0.47 g L⁻¹ h⁻¹, indicating that the enzymatic hydrolysates may contain stimulating compounds that can improve the ABE fermentation.     

Performances comparison between three technologies for continuous ethanol production from molasses

Molasses are a potential feedstock for ethanol production. The successful application of anaerobic fermentation for ethanol production from molasses is critically dependent to the development and the use of high rate bioreactors. In this study the fermentation of sugar cane molasses by Saccharomyces cerevisiae for the ethanol production in a continuously stirred tank reactor (CSTR), an immobilised cell reactor (ICR) and a membrane reactor (MBR) was investigated. Ethanol production and reactor productivities were compared under different dilution rates (D). When using the CSTR, a decent ethanol productivity (Qp) of 6.8 g L⁻¹ h⁻¹ was obtained at a dilution rate of 0.5 h⁻¹. The Qp was improved by 48% and the residual sugar concentration was reduced by using the ICR. Intensifying the production of ethanol was investigated in the MBR to achieve a maximum ethanol concentration and a Qp of 46.5 g L⁻¹ and 19.2 g L⁻¹ h⁻¹, respectively. The achieved results in the MBR worked with high substrate concentration are promising for the scale up operation.     

Lipid accumulation in the new oleaginous yeast Debaryomyces etchellsii correlates with ascosporogenesis

The growth characteristics, lipid accumulation and composition during the life cycle of a newly isolated strain of Debaryomyces etchellsii were studied under nitrogen limiting conditions. This yeast, grown in batch flask or bioreactor cultures, reproduced asexually by buds when nitrogen was available in the growth medium, or sexually by ascospores after nitrogen exhaustion, producing more than 7 g L⁻¹ biomass. During ascosporogenesis, an important increase in the cellular lipid content in dry cell mass occurred, i.e. from a mass fraction of 11.9% in the vegetative phase to 22.4%, in the ascosporogenic phase. During transition of D. etchellsii from batch to continuous cultures using dilution rates 0.026 and 0.019 h⁻¹, a shift from sexual to asexual reproduction was observed. At 0.019 h⁻¹, few pseudomycelia were also formed. The yeast synthesized lipids containing long chain fatty acids (mainly C16 and C18). Budded cells at steady-states contained only 8.6–9.3 % of lipids mass fraction per dry cell mass that were composed of oleic and linoleic acids and, to a lesser extent, of palmitic and palmitoleic acids. Neutral lipids were the major fraction represented 61.8–66.1%, of total lipids followed by phospholipids, which was the only fraction in which linoleic acid predominated over oleic acid.     

Prospecting the biofuel potential of new microalgae isolates

The continued search and urgent need for renewable fuel sources have necessitated the exploration of microalgae to identify relevant species for making biofuels. The aim of the study was bioprospecting and screening native microalgae strains from freshwater habitats of the Almaty region, Kazakhstan, to assess the potential for producing biofuel. The studied strains demonstrated simultaneous biomass productivity, lipid productivity, suitable fatty acid composition, and biodiesel properties. The sequence analysis of the ribosomal DNA internal transcribed spacer partial region and ribulose-bisphosphate carboxylase gene (rbcL) led to the identification of five microalgae: Monoraphidium griffithii ZBD-01, Nephrochlamys subsolitaria ZBD-02, Ankistrodesmus falcatus ZBD-03, Parachlorella kessleri ZBD-04, and Desmodesmus pannonicus ZBD-05. P. kessleri had the highest biomass production (1.42 ± 0.08 g L⁻¹ day⁻¹), lipid productivity (29 ± 1.2 g L⁻¹day⁻¹), and C₁₆–C₁₈ fatty acid contents (90%), followed by A. falcatus and M. griffithi. Gas chromatography/mass spectrometry analysis indicated that the dominant fatty acids in these strains were palmitic, stearic, and oleic acids. The calculated biodiesel properties of P. kessleri and A. falcatus based on fatty acid methyl esters (FAME) profiles showed relatively good fuel properties (cetane numbers - 48 and 50; iodine and saponification values - 83.4 and 103.6 g I₂/100 g oil, 260.8 and 199.5 mg KOH g⁻¹), which correlate well with. Our results suggest that P. kessleri and A. falcatus are promising strains for biodiesel production due to their high lipid productivity, fatty acid profile with relatively high content of oleic acid, and suitable biodiesel properties. The isolated native species of microalgae from natural freshwater bodies of the Almaty region present opportunities for further exploitation for the sustainable production of biomass and biodiesel.     

Statistical optimization of physical process variables for bio-plastic (PHB) production by Alcaligenes sp.

In the present study, efforts have been made to optimize the three physical process variables viz; pH, temperature and agitation speed for enhanced polyhydroxybutyrate (PHB) production in batch cultivation by Alcaligenes sp. which serves as precursor for bio-plastic (PHB) production. Strain selection was done by viable staining method using nile blue A dye. Agro-industrial by products; cane molasses and urea were used as carbon and nitrogen source for PHB production. Optimization of physical process variables was done by central composite rotatable design (CCRD) using design expert (DX 8.0.6) software. Shake flask cultivation performed under optimum physical condition viz; 34.5 °C temperature, 6.54 pH and agitation speed of 3.13 Hz, gave PHB mass fraction yield of 76.80% on dry molasses substrate and showed 98.0% resemblance with the predicted percentage yield of 77.78%. Batch cultivation further performed in 7.5 L lab scale bioreactor (working volume: 5.6 L) under optimized condition gave maximum cell biomass of 11 ± 0.5 g L⁻¹ with a PHB content of 8.8 ± 0.4 g L⁻¹ after 48.0 h of fermentation. Scale up study on bioreactor gave maximum PHB yield (Y_P/x) and productivity of 0.78 and 0.19 g L⁻¹ h, which are higher than previous reports under similar condition. Characterization of PHB was done by FTIR.     

The kinetics of Scenedesmus obliquus microalgae growth utilizing carbon dioxide gas from biogas

Microalgae Scenedesmus obliquus was cultured in a laboratory photobioreactor to determine the efficacy of using biogas as a carbon source for the microalgae's growth. The biogas contained ∼60% CH₄ and ∼40% CO₂, and was derived from an anaerobic digester operating from animal wastes, and an anaerobic reactor utilizing high strength wastewater. The results showed that biogas is a viable carbon source for microalgae growth and that significant portions of the biogas' CO₂ can be utilized for algae growth, resulting in a biogas having a high concentration of methane. This paper develops the kinetic expressions for the algae's growth by assuming an autocatalytic reaction between carbon substrate and microalgae. The maximum specific growth rate and biomass productivity of S. obliquus were 0.56 d⁻¹ and 0.145 g L⁻¹d⁻¹ respectively. The biomass contained 51.8% carbon and higher heating value (HHV) was 22.9 MJ kg⁻¹.     

Significance of carbon to nitrogen ratio on the long-term stability of continuous photofermentative hydrogen production

The stable and optimized operation of photobioreactors (PBRs) is the most challenging task in photofermentative biological hydrogen production. The carbon to nitrogen ratio (C/N) in the feed is a critical parameter that significantly influences microbial growth and hydrogen production. In this study, the effects of changing the C/N ratio to achieve stable biomass and continuous hydrogen production using fed-batch cultures of Rhodobacter capsulatus YO3 (uptake hydrogenase deleted, hup-) were investigated. The experiments were carried out in 8 L panel PBRs operated in indoor conditions under continuous illumination and controlled temperature. Culture media containing different acetate (40-80 mM) and glutamate (2-4 mM) concentrations were used to study the effects of changing the C/N ratio on biomass growth and hydrogen production. Stable biomass concentration of 0.40 g dry cell weight per liter culture (gDCW/L_c) and maximum hydrogen productivity of 0.66 mmol hydrogen per liter culture per hour (mmol/L_c/h) were achieved during fed-batch operation with media containing 40 mM acetate and 4 mM glutamate, C/N = 25, for a period of over 20 days. A study on the effect of biomass recycling on biomass growth and hydrogen production showed that the feedback of cells into the photobioreactor improved biomass stability during the fed-batch operation but decreased hydrogen productivity.