Isolation and characterization of a novel hydrogen-producing strain Clostridium sp. suitable for immobilization

A hydrogen-producing strain of bacteria suitable for immobilization was isolated from anaerobic sludge obtained from a methane fermentation plant. The isolated strain, CFPA-20 was identified as a novel species of the genus Clostridium by phylogenetic analysis of the 16S rRNA sequence. The changes in free energy of interaction of adhesion to polymer resin and self-aggregation were both negative. This indicated that CFPA-20 was thermodynamically favored for immobilization. CFPA-20 grew at a temperature range of 25–37 °C and at a pH range of 4.5–9.0. Immobilization of CFPA-20 on block copolymer polyethylene glycol-b-polypropylene glycol gave a radically improved hydrogen production yield (2.91 mol/mol-glucose) and a maximum hydrogen production rate (568 mL/L-culture/h) compared to the non-immobilized isolate. In addition, the biofilm of CFPA-20 acquired tolerance for volatile fatty acids. Further investigation into this mechanism may ultimately improve the hydrogen production capacity of CFPA-20.     

Vegetable oil production potential from Jatropha curcas, Croton megalocarpus, Aleurites moluccana, Moringa oleifera and Pachira glabra: Assessment of renewable energy resources for bio-energy production in Africa

Research on vegetable oil for biofuels in Africa and Asia has focused mainly on Jatropha curcas while other potential oil bearing plants have received little attention. Vegetable oil production potential for five oil bearing plant species namely: Aleurites moluccana, Croton megalocarpus, Jatropha curcas, Moringa oleifera and Pachira glabra were investigated. Nuts and seeds of the plants were collected from the wild and their potential for vegetable oil production assessed in terms of seed/nut acreage yield, seed/nut oil content, harvesting requirement, and upstream processing before vegetable oil recovery. All five varieties were found to contain acceptable but different oil content ranging from 20 to 33% w/w, and seed/nut acreage yield of 3 t ha⁻¹ y⁻¹ to 12.5 t ha⁻¹ y⁻¹. Upstream processing was needed for A. moluccana to break open nuts to release the kernel, and dehulling for both C. megalocarpus and J. curcas to release the seeds, before extracting the vegetable oil, while the seeds of both M. oleifera and P. glabra did not need upstream processing. The Multi-criteria Decision Analysis ranked C. megalocarpus as the plant with the highest vegetable oil production potential of 1.8 t ha⁻¹ y⁻¹ followed by M. oleifera, J. curcas (1 t ha⁻¹ y⁻¹), A. moluccana, and P. glabra. The analysis underlines the need for more studies on C. megalocarpus and M. oleifera for biofuel production in Africa and other regions.     

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.     

Isochrysis sp. IOAC724S,a newly isolated,lipid-enriched,marine microalga for lipid production,and optimized cultivation conditions

An oleaginous, unicellular, marine microalga termed IOAC724S was isolated from the South China Sea. Morphology and genetic analyses indicated it belongs to the genus Isochrysis. Gas chromatography (GC) results showed that more than 10 types of fatty acids existed in Isochrysis sp. IOAC724S and that 90% of them were suitable for lipid production. The culture conditions suitable for cell growth were progressively optimized through photosynthetic and respiratory analyses. The optimal culture conditions were: photon flux 200–500 μmol m⁻² s⁻¹, temperature 35 °C during daytime and 24 °C at night, pH value between 7 and 8, NaNO₃ 160 g m⁻³ and NaH₂PO₄·2H₂O 80 g m⁻³ for starting culture. When microalgal cultures were exposed to these optimal conditions, the specific growth rate reached to 0.26 d⁻¹ on average and 1.0 d⁻¹ in MAX. Lipid production was optimized through nutrient starvation processes, including nitrate or phosphate deprivation and simultaneous nitrate and phosphate deprivation. The highest lipid mass fraction of dry cell weight (about 55.6%) was obtained after the stationary phase algal culture was transferred into phosphate-free medium for 3 days. GC data demonstrated that the enhancement of lipid accumulation in algal cells maintained under nutrient starvation came mainly from an increase of C16:0 and C18:1 fatty acids; however, the lipids with a chain length appropriate for fuel use (C14 to C18) were unchanged at 90% mass fraction of the dry cell weight. Based on these good characteristics, Isochrysis sp. IOAC724S appeared to be a strong candidate for lipid production.     

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.     

Biodiesel production using gel‐type cation exchange resin at different ionic forms

In this study, a strong acidic‐type cation exchange resin was used in the transesterification of corn oil to fatty acid methyl esters (FAME). The gel‐type cation exchange resin (Purolite‐PD206) was used in H⁺ and Na⁺ forms to utilize ion‐exchange resin as effective heterogeneous catalyst in the production of biodiesel. Effect of ionic forms of ion exchange resin on free fatty acid (FFA) conversion and composition was investigated by using different amounts of ion exchange resin (12, 16, and 20 wt%), various mole ratios of methanol to oil (1:6, 1:12, and 1:18 mol/mol), reaction temperatures (63, 65, and 67°C), and reaction time (24, 36, and 48 h) during transesterification reaction. The highest FFA conversions of 73.5% and 79.45% were obtained at conditions of 20 wt% of catalyst, 65°C of reaction temperature, 18:1 as methanol to oil ratio, and 48 h of reaction time for H⁺ and Na⁺ forms of ion exchange resin, respectively. These results were obtained from regression equations established by using analysis of variance (ANOVA) model according to the experimental results of selected parameters. Gas chromatography analysis revealed that FAME is mainly composed of C16:0 (palmitic), C18:1 (oleic), and C18:2 (linoleic) acids of methyl ester.     

Biodiesel production by esterification of palm fatty acid distillate

Production of fatty acid methyl ester (FAME) from palm fatty acid distillate (PFAD) having high free fatty acids (FFA) was investigated in this work. Batch esterifications of PFAD were carried out to study the influence of: including reaction temperatures of 70–100 °C, molar ratios of methanol to PFAD of 0.4:1–12:1, quantity of catalysts of 0–5.502% (wt of sulfuric acid/wt of PFAD) and reaction times of 15–240 min. The optimum condition for the continuous esterification process (CSTR) was molar ratio of methanol to PFAD at 8:1 with 1.834 wt% of H₂SO₄ at 70 °C under its own pressure with a retention time of 60 min. The amount of FFA was reduced from 93 wt% to less than 2 wt% at the end of the esterification process. The FAME was purified by neutralization with 3 M sodium hydroxide in water solution at a reaction temperature of 80 °C for 15 min followed by transesterification process with 0.396 M sodium hydroxide in methanol solution at a reaction temperature of 65 °C for 15 min. The final FAME product met with the Thai biodiesel quality standard, and ASTM D6751-02.     

Biosorptive removal of Pband Cd onto novel biosorbent: Defatted Carica papaya seeds

Carica papaya seeds, an agricultural waste in Nigeria, were defatted to obtain defatted C. papaya seed biosorbent. The Fourier Transformed Infrared spectrum of defatted C. papaya seed biosorbent suggests the presence of CO, OH of carboxylic acid, lactonic and amide band functional groups. The adsorption of metal ion onto defatted C. papaya seed biosorbent led to small shifts in the IR bands. The adsorption capacity of defatted C. papaya seed biosorbent was evaluated to be 1666.67 mg/g for Pb²⁺ and 1000.00 mg/g for Cd²⁺. In binary metal ion solution, the defatted C. papaya seeds showed decreased adsorption capacity for either metal ion. The influence of different particle sizes was found to have negative impact on the adsorption capacity of C. papaya seed biosorbent in the removal of Pb²⁺ and Cd²⁺ from aqueous solution. The adsorption of both metal ions was observed to follow the Freudlich model better than the Langmuir model suggesting that the adsorption of both metal ions was on multisites on the defatted C. papaya seed biosorbent. The adsorption was found to be highly feasible, spontaneous and exothermic in nature. Optimization results suggests 5 m³ of 100 mg/L of Pb²⁺ and Cd²⁺ requires 43.3 and 49.2 kg of defatted C. papaya seeds to remove 95% of the metal ions from aqueous solution.     

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.     

Biodiesel from Milo (Thespesia populnea L.) seed oil

There is a need to seek non-conventional seed oil sources for biodiesel production due to issues such as supply and availability as well as food versus fuel. In this context, Milo (Thespesia populnea L.) seed oil was investigated for the first time as a potential non-conventional feedstock for preparation of biodiesel. This is also the first report of a biodiesel fuel produced from a feedstock containing cyclic fatty acids as T. populnea contains 8,9-methylene-8-heptadecenoic (malvalic) and smaller amounts of two cyclopropane fatty acids besides greater amounts of linoleic, oleic and palmitic acids. The crude oil extracted from T. populnea seed was transesterified under standard conditions with sodium methoxide as catalyst. Biodiesel derived from T. populnea seed oil exhibited fuel properties of density 880 kg m⁻³, kinematic viscosity 4.25 mm²/s; cetane number 59.8; flash point 176 °C; cloud point 9 °C; pour point 8 °C; cold filter plugging point 9 °C; sulfur content 11 mg kg⁻¹; water content 150 mg kg⁻¹; ash content 15 mg kg⁻¹; and acid value as KOH 250 mg kg⁻¹. The oxidative stability of 2.91 h would require the use of antioxidants to meet specifications in standards. Generally, most results compared well with ASTM D6751 and EN 14214 specifications.     

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.     

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.     

Harnessing of biohydrogen by acidogenic fermentation of Citrus limetta peelings: Effect of extraction procedure and pretreatment of biocatalyst

The extracts of Citrus limetta (sweet lime) peelings were evaluated as a fermentable substrate for hydrogen (H₂) production by dark-fermentation (acidogenic) using both anaerobic mixed consortia and selectively enriched acidogenic mixed consortia. Extraction was carried by pretreating sweet lime peelings at 121 °C (1 bar pressure) at variable pH (6 and 7) and digestion time (20 and 40 min). Maximum organic matter extraction was observed at pH 7.0 (40 min). Fermentation was performed at different organic loading conditions [OL1, 1.17 kg COD/m³; OL2, 2.35 kg COD/m³; OL3, 4.69 kg COD/m³] under acidophilic microenvironment. H₂ production was found to depend on the concentration of the substrate and composition. Increase in organic load showed consistent improvement in H₂ production. Operation at OL3 employing selectively enriched inoculum documented higher cumulative H₂ production (10.07 mmol) and H₂ production rate (0.345 mmol/h) (pH 7; 40 min). Substrate degradation was also found to increase with increase in organic loading. Maximum substrate degradation (SD) was registered at pH 6 (40 min) with anaerobic culture (2.80 kg COD_R/m³; ξ_COD 31.82%) and at pH 7 (40 min) with selectively enriched acidogenic culture (3.20 kg COD_R/m³; ξ_COD 36.36%). Concentration of volatile fatty acids (VFAs) also improved with increase in organic load. Maximum VFA concentration (1098 mg/l) was observed with OL3 (pH 7; 40 min) by using selectively enriched culture.     

Heterologous expression and production of Trichoderma reesei cellobiohydrolase II in Pichia pastoris and the application in the enzymatic hydrolysis of corn stover and rice straw

The cellobiohydrolase II (CBH II) gene cbh2 from Trichoderma reesei was cloned and its codons were optimized in accordance with the codon usage frequencies of the host Pichia pastoris. The AOX1 strong promoter inducible by methanol was employed to efficiently express the foreign gene cbh2 in P. pastoris. It was found that 5.84 ± 0.42 U cm⁻³ CBH II was obtained at 96 h using the synthetic cbh2 gene whose codons were optimized, 2.02-fold higher than using the native cbh2 gene (2.89 ± 0.32 U cm⁻³), indicating that the codon optimization strategy was an effective approach to enhance the heterologous expression of CBH II in P. pastoris. The product of recombinant P. pastoris CBH II had an approximate molecular weight 58 kDa. Its optimal pH and temperature were 5.0 and 50 °C, respectively. The recombinant CBH II was used to enhance the yields of the enzymatic hydrolysis of the corn stover and rice straw pretreated with sodium hydroxide by improving the exo-exo-synergism between CBH II and CBH I in T. reesei cellulase. The yields 94.7% and 83.3% were achieved in the enzymatic hydrolysis of corn stover and rice straw pretreated by sodium hydroxide, respectively.     

Enhanced hydrogen production from biomass via the sulfur redox cycle under hydrothermal conditions

A new method of hydrogen production from biomass via a sulfur redox cycle at moderate temperatures has been proposed. This method, which can utilize excess sulfur from hydrocarbon refining processes and waste or geothermal heat, consists of two half cycles: (1) hydrogen production from an aqueous alkaline solution at subcritical conditions of water, where sulfide, HS⁻ and S²⁻, acts as a reducing agent of water, and (2) sulfide regeneration under much milder conditions, with an organic compound derived from biomass acting as a reducing agent of polysulfide, S_n²⁻, and sulfur oxyanion, S_xO_y²⁻, formed in the first half cycle. During a 60-min reaction of an aqueous sodium sulfide solution, hydrogen production was observed at ≥280 °C and corresponding saturated vapor pressures. Addition of D-glucose, C₆H₁₂O₆, to the solution after hydrogen production at 300 °C resulted in sulfide regeneration at temperatures ≥60 °C in the present 10-min reaction. Moreover, hydrogen production from glucose via the sulfur redox cycle was demonstrated, where the hydrogen production and sulfide regeneration were conducted at 300 °C and 105 °C, respectively. Results indicated that hydrogen production from 1 mol glucose was greater than that by hydrothermal gasification of glucose at much higher temperatures up to 500 °C.     

Mesophilic biohydrogen production by Clostridium butyricum CWBI1009 in trickling biofilter reactor

This study investigates the mesophilic biohydrogen production from glucose using a strictly anaerobic strain, Clostridium butyricum CWBI1009, immobilized in a trickling bed sequenced batch reactor (TBSBR) packed with a Lantec HD Q-PAC^® packing material (132 ft²/ft³ specific surface). The reactor was operated for 62 days. The main parameters measured here were hydrogen composition, hydrogen production rate and soluble metabolic products. pH, temperature, recirculation flow rate and inlet glucose concentration at 10 g/L were the controlled parameters. The maximum specific hydrogen production rate and the hydrogen yield found from this study were 146 mmol H₂/L.d and 1.67 mol H₂/mol glucose. The maximum hydrogen composition was 83%. Following a thermal treatment, the culture was active without adding fresh inoculum in the subsequent feeding and both the hydrogen yield and the hydrogen production rate were improved. For all sequences, the soluble metabolites were dominated by the presence of butyric and acetic acids compared to other volatile fatty acids. The results from the standard biohydrogen production (BHP) test which was conducted using samples from TBSBR as inoculum confirmed that the culture generated more biogas and hydrogen compared to the pure strain of C. butyricum CWBI1009. The effect of biofilm activity was studied by completely removing (100%) the mixed liquid and by adding fresh medium with glucose. For three subsequent sequences, similar results were recorded as in the previous sequences with 40% removal of spent medium. The TBSBR biofilm density varied from top to bottom in the packing bed and the highest biofilm density was found at the bottom plates. Moreover, no clogging was evidenced in this packing material, which is characterized by a relatively high specific surface area. Following a PCA test, contaminants of the Bacillus genus were isolated and a standard BHP test was conducted, resulting in no hydrogen production.     

Biohydrogen production from algal biomass (Anabaena sp. PCC 7120) cultivated in airlift photobioreactor

The present study deals with the optimization of pretreatment conditions followed by thermophilic dark fermentative hydrogen production using Anabaena PCC 7120 as substrate by mixed microflora. Different airlift photobioreactors with ratio of area of downcomer and riser (A_d/A_r) in range of 0.4–3.2 were considered. Maximum biomass concentration of 1.63 g L⁻¹ in 9 d under light intensity of 120 μE m⁻² s⁻¹ was observed at A_d/A_r of 1.6. The mixing time of the reactors was inversely proportional to A_d/A_r. Maximal H₂ production was found to be 1600 mL L⁻¹ upon pretreatment with amylase followed by thermophilic fermentation for 24 h compared to other methods like sonication (200 mL L⁻¹), autoclave (600 mL L⁻¹) and HCl treatment (1230 mL L⁻¹). The decrease of pH from 6.5 to 5.0 during fermentation was due to the accumulation of volatile fatty acids. Amylase pretreatment gave higher reducible sugar content of 7.6 g L⁻¹ as compare to other pretreatments. Thermophilic fermentation of pretreated Anabaena biomass by mixed bacterial culture was found suitable for H₂ production.     

Optimization of culture conditions and electricity generation using Geobacter sulfurreducens in a dual-chambered microbial fuel-cell

The promise of generating electricity from the oxidation of organic substances using metal-reducing bacteria is drawing attention as an alternate form of bio-technology with positive environmental implications. In this study, we examined various experimental factors to obtain the maximum power output in a dual-chamber mediator-less microbial fuel-cell (MFC) using Geobacter sulfurreducens and acetate as an electron donor in a semi-continuous mode. The G. sulfurreducens culture conditions were optimized in a nutrient buffer containing 20 mM of acetate and 50 mM of fumarate at pH 6.8 and 30 °C. For use in the MFC system, electrodes were made with carbon paper (area: 11.5 cm²) and spaced 1.5 cm apart. Once the MFC was inoculated with the pre-cultured G. sulfurreducens in the anode chamber and while air was continuously sparged to the cathode chamber, the cells produced electricity stably over 60 days with the regular addition of 20 mM acetate, generating the maximum power density of 7 mW/m² with a 5000 Ω load. The current output was significantly increased, by 1.6 times after 20 days of incubation under the same experimental conditions, when the carbon-paper anode was coated with carbon nanotubes.     

Influence of inoculum cell density and carbon dioxide concentration on fed-batch cultivation of Nannochloropsis oculata

The marine microalgae Nannochloropsis oculata is a promising source of biofuel because of its high lipid content. For achieving high productivity of oil from microalgae, a high cell concentration before harvesting is beneficial. The present study investigated fed-batch cultures of N. oculata fed with vitamins and nutrient solutions and found that the biomass yield of N. oculata in the fed-batch culture was 1.25 times higher than that in batch culture. Fed-batch cultivation, especially at high illumination, decreased the inhibitory effect of high carbon dioxide (CO₂) concentration on the microalgal growth. The specific growth rate was directly proportional to the light intensity in the CO₂ environment. A light intensity of 40,000 Lux was able to achieve high specific growth rates in fed-batch cultivation at a CO₂ volume fraction of 2%–15%. The tolerance of N. oculata to CO₂ was enhanced by the daily feeding of nutrients in the fed-batch cultivation. At 2% CO₂, a final cell density of about OD₆₈₂ = 11.4 was achieved in the fed-batch culture in 30 days. Furthermore, a cell density of 14.4 g L⁻¹ was obtained by outdoor fed-batch cultivation in 27 days.     

Optimization of temperature and light intensity for improved photofermentative hydrogen production using Rhodobacter capsulatus DSM 1710

Photofermentative hydrogen production is influenced by several parameters, including feed composition, pH levels, temperature and light intensity. In this study, experimental results obtained from batch cultures of Rhodobacter capsulatus DSM 1710 were analyzed to locate the maximum levels for the rate and yield of hydrogen production with respect to temperature and light intensity. For this purpose, a 3^k general full factorial design was employed, using temperatures of 20, 30 and 38 °C and light intensities of 100, 200 and 340 W/m². ANOVA results confirmed that these two parameters significantly affect hydrogen production. Surface and contour plots of the regression models revealed a maximum hydrogen production rate of 0.566 mmol H₂/L/h at 27.5 °C and 287 W/m² and a maximum hydrogen yield of 0.326 mol H₂/mol substrate at 26.8 °C and 285 W/m². Validation experiments at the calculated optima supported these findings.