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.     

Improvement in energy recovery from Chlorella sp. biomass by integrated dark-photo biohydrogen production and dark fermentation-anaerobic digestion processes

Chlorella sp. biomass was used as the sole substrate for the production of hydrogen and methane through integrated dark fermentation (DF) and photo-fermentation (PF), and DF and anaerobic digestion (AD) processes. Prior to use in fermentations, the biomass was pretreated by acid-hydrothermal method, which yielded a maximum reducing sugar yield of 162.9 ± 4.2 mg g-biomass⁻¹. The use of the microalgal hydrolysate to produce hydrogen by DF gave a hydrogen yield (HY) of 47.2 ± 1.1 mL g-volatile-solids⁻¹ (VS). The subsequent use of the hydrogenic effluent in PF gave a HY of 125.0 ± 1.5 mL g-VS⁻¹, while AD of the hydrogenic effluent gave a methane yield of 152.8 ± 1.3 mL g-VS⁻¹. The total energy yield attained by the use of DF alone, the integrated DF-PF, and DF-AD processes were 0.51, 1.86 and 5.98 kJ g-VS⁻¹, respectively. These results indicate that the integrated DF-AD process was effective in recovering energy from Chlorella sp. biomass. However, an energy balance analysis indicated that the process was not energetically feasible due to the high energy demand for the acid-hydrothermal pretreatment.     

Improvement of biohythane production from Chlorella sp. TISTR 8411 biomass by co-digestion with organic wastes in a two-stage fermentation

The suitability of molasses, Napier grass (Pennisetum purpureum), empty fruit bunches (EFB), palm oil mill effluent (POME), and glycerol waste as a co-substrate with Chlorella sp. TISTR 8411 biomass for biohythane production was investigated. Mono-digestion of Chlorella biomass had hydrogen and methane yield of 23–35 and 164–177 mL gVS⁻¹, respectively. Co-digestion of Chlorella biomass with 2–6% TS of organic wastes was optimized for biohythane production with hydrogen and methane yield of 17–75 and 214–577 mL gVS⁻¹, respectively. The hydrogen and methane yield from co-digestion of Chlorella biomass with molasses, POME, and glycerol waste was increased by 8–100% and 80–264%, respectively. The biohythane production of co-digestion of Chlorella was 6–11 L L-mixed waste⁻¹ with an optimal C/N ratio range of 19–41 and H₂/CH₄ ratio range of 0.06–0.3. Co-digestion of Chlorella biomass was significantly improved biohythane production in term of yield, production rate, and kinetics.     

Bio-hythane production from residual biomass of Chlorella sp. biomass through a two-stage anaerobic digestion

Residual Chlorella sp. biomass obtained after anaerobic solid-state fermentation was used to produce bio-hythane. The residual biomass was pretreated using acid, thermal, and acid-thermal methods before their respective hydrolysates were used in dark fermentation followed by the methanogenesis of anaerobic digestion to produce hydrogen and methane, respectively. Pretreatment of the residual biomass using acid and thermal methods did not significantly increase reducing sugar production. However, a maximum reducing sugar content of 28.9 mg-reducing-sugar·g-biomass⁻¹ was attained using an acid-thermal method, resulting in the highest hydrogen and methane yields of 12.5 and 81 mL·g-volatile-solid⁻¹, respectively. This was equivalent to the total energy yield of 3.03 kJ·g-VS⁻¹ or 4.6% energy recovery, based on the heating value of the residual biomass.     

Biohydrogen production by novel cyanobacterial strains isolated from rice paddies in Kazakhstan

A limited supply of oil prompts the search for non-traditional energy sources to replace traditional ones. This makes hydrogen gas an appealing alternative source. Photosynthetic organisms capture sunlight very efficiently and convert it into organic molecules. A promising wild strain was isolated for the first time, from the rice paddies of Kazakhstan (Kyzylorda and Almaty regions), which can be considered as one of the most active hydrogen producers compared to the literature. The result showed that among the 13 isolated and collection cyanobacterial strains, Synechocystis sp. S-1 is the most active H₂ producer (2.35 μmol H₂ mg^?1 Chl a h^?1) in the light. In contrast, the wild-type cyanobacterium Anabaena variabilis A-1 had higher productivity, nitrogenase activity, and a stronger capacity to produce hydrogen in the dark (8.67 μmol H₂ mg^?1 Chl a h^?1), which matched the maximum yield obtained in the research. The metabolic modulation performed significantly increased hydrogen production. The highest photohydrogen production rate was observed in cells incubated with 25 μmol HEPES and 50 μmol sodium bicarbonate (NaHCO₃).     

Coupling algal biomass production and anaerobic digestion: Production assessment of some native temperate and tropical microalgae

Coupling algal biomass production and anaerobic digestion is one of the most promising bioprocesses for economically viable algal production. This study assesses the production rates of some native microalgae growing in media supplemented with algal digestate, urban wastewater or digested sludge. Native microalgal populations isolated from temperate freshwaters (Scenedesmus spp.) and marine ecosystems (Nannochloris spp.) had the highest potential production rates (about 100 mg DW L⁻¹ d⁻¹) with algal digestate at about 20% loading ratio. However, no growth was measured for Nannochloris spp., when the ammonium concentration exceeded 100 mg L⁻¹ although Scenedesmus spp. appeared to be tolerant to higher NH₄⁺ concentrations. Very low production rates, or no growth, were measured when microalgae isolated from high salinity waters (Dunaliella salina, Lyngbya aestuarii) were used, suggesting that populations well adapted to extreme environmental conditions are not suitable candidates for growing on wastewater or anaerobic digestate.     

Characterization,pretreatment and saccharification of spent seaweed biomass for bioethanol production using baker's yeast

Seaweeds are marine macroalgae found abundantly and viewed as potential source of phycocolloids to produce biofuel. In this study, seaweed spent biomass obtained from alginate production industry and biomass obtained after pigment extraction were found to contain a considerable amount of phycocolloids. These two spent biomasses were investigated for the production of ethanol. In this study, the red seaweed spent biomass of Gracilaria corticata var corticata showed higher content of polysaccharide (190.71 ± 30.67 mg g⁻¹ dry weight) than brown seaweed spent biomass (industrial) (136.28 ± 30.09 mg g⁻¹ dry weight). Hydrolysis of spent biomasses with different concentrations of sulfuric acid (0.1%, 0.5% and 1%) was also investigated. Brown seaweed spent biomass and red seaweed spent biomass exhibited high amount of sugar in 0.5% and 1% sulfuric acid treatment, respectively. Proximate and ultimate composition of seaweed spent biomasses were analysed for energy value. The FT-Raman spectra exhibited similar stretches for both acid hydrolysed spent biomasses with their respective standards. Ethanol produced through a fermentation process using spent hydrolysates with baker's yeast at pH 5.3 was found to be significant. The ethanol yield from brown seaweed spent biomass and red seaweed spent biomass was observed to be 0.011 g g⁻¹ and 0.02 ± 0.003 g g⁻¹ respectively, when compared with YPD (0.42 ± 0.03 g g⁻¹) and d-galactose (0.37 ± 0.04 g g⁻¹) as standard on day 4. The present study revealed the possibility of effective utilization of spent biomass from seaweed industry for ethanol production.     

High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition

Biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis was investigated under thermophilic condition. The optimum chemical oxygen demand (COD) concentration and pH for dark fermentation were 66 g·L⁻¹ and 6.5 with a hydrogen yield of 73 mL-H₂·gCOD⁻¹. The dark fermentation effluent consisted of mainly acetate and butyrate. The optimum voltage for microbial electrolysis was 0.7 V with a hydrogen yield of 163 mL-H₂·gCOD⁻¹. The hydrogen yield of continuous two-stage dark fermentation and microbial electrolysis was 236 mL-H₂·gCOD⁻¹ with a hydrogen production rate of 7.81 L·L⁻¹·d⁻¹. The hydrogen yield was 3 times increased when compared with dark fermentation alone. Thermoanaerobacterium sp. was dominated in the dark fermentation stage while Geobacter sp. and Desulfovibrio sp. dominated in the microbial electrolysis cell stage. Two-stage dark fermentation and microbial electrolysis under thermophilic condition is a highly promising option to maximize the conversion of palm oil mill effluent into biohydrogen.     

Trace metals supplementation enhanced microbiota and biohythane production by two-stage thermophilic fermentation

The effect of trace metals supplementation into palm oil mill effluent on biohythane production and responsible microbial communities in thermophilic two-stage anaerobic fermentation was investigated. High biohythane yields were linked to Ni/Co/Fe supplementation (10, 6 and 20 mg L⁻¹, respectively) with maximum H₂ and CH₄ yields of 139 mL H₂ gVS⁻¹ and 454 mL CH₄ gVS⁻¹, respectively. The Ni/Co/Fe supplementation resulted in higher numbers of Bacillus sp., Clostridium sp. and Thermoanaerobacterium sp. together with increasing hydrogenase expression level leading to increasing hydrogen yields of 90.4%. The numbers of Methanosarcina, Methanomassiliicoccus, and Methanoculleus were enhanced by Ni/Co/Fe addition, accompanied by 21.7% higher methane yields. No correlation between methyl coenzyme-M reductase expression level and methane yields was observed. The Ni/Co/Fe supplementation improved gas production in the two-stage biohythane process via enhancing a number of viable hydrogen-producing bacteria together with hydrogenase activity in H₂ stage and enhancing number methanogens in the CH₄ stage.     

Enhancement of hydrogen production by the filamentous non-heterocystous cyanobacterium Arthrospira sp. PCC 8005

The efficiency of hydrogen production by different cyanobacterial species depends on several external factors. We report here the factors enhancing hydrogen production by filamentous non-heterocystous cyanobacterium Arthrospira sp. PCC 8005. Cells adapted to dark-anaerobic conditions produced hydrogen consistent with increased hydrogenase activity when supplemented with Fe²⁺. Stimulation of hydrogen production could be achieved by addition of reductants, either dithiothreitol or β-mercaptoethanol with higher production observed with the latter. Additionally, Fe²⁺ and β-mercaptoethanol added to nitrogen- and sulphur-deprived cells significantly stimulated H₂ production with maximal value of 5.91 ± 0.14 μmol H₂ mg Chla⁻¹ h⁻¹. Glucose and a small increase of osmolality imposed by either NaCl or sorbitol enhanced hydrogen production. High rates of hydrogen production were obtained in cells adapted in nitrogen-deprived medium with neutral and alkaline external pH, significant decrease of hydrogen production occurred under acidic external pH.     

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.     

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.     

From piggery wastewater nutrients to biogas: Microalgae biomass revalorization through anaerobic digestion

Microalgae grown in swine wastewater were used as a promising strategy to produce renewable energy by coupling wastewater bioremediation and biomass revalorization. The efficiency of a microalgae consortium treating swine slurry at different temperature (15 and 23 °C) and illumination periods (11 and 14 h) was assessed for biomass growth and nutrient removal at two NH₄⁺ initial concentrations (80 and 250 mg L⁻¹ NH₄⁺). Favourable culture conditions (23 °C and 14 h of illumination) and high ammonium loads resulted in higher biomass production and greater nutrients removal rates. The initial N–NH₄⁺ load determined the removal mechanism, thus ammonia stripping and nitrogen uptake accounted similarly in the case of high NH₄⁺ load, while nitrogen uptake prevailed at low NH₄⁺ load. Under favourable conditions, nitrogen availability in the media determined the composition of the biomass. In this context, carbohydrate-rich biomass was obtained in batch mode while semi-continuous operation resulted in protein-rich biomass. The revalorization of the resultant biomass was evaluated for biogas production. Methane yields in the range of 106–146 and 171 ml CH₄ g COD⁻¹ were obtained for the biomasses grown in batch and semi-continuous mode, respectively. Biomass grown under favourable conditions resulted in higher methane yields and closer to the theoretically achievable.     

Screening and optimisation of hydrogen production by newly isolated nitrogen-fixing cyanobacterial strains

Recently, there has been a propensity to postpone dealing with the world's climate concerns until later, resulting in a 1.5 °C rise in temperature over the last century. Therefore, interest in biologically derived, inexhaustible energy sources based on solar energy is growing. Cyanobacteria have the potential to produce clean, renewable fuels in the form of hydrogen (H₂) gas, derived from solar energy and water. The current study reports the screening 11 cyanobacterial strains isolated from rice paddies and hotsprings for efficient H₂ producers. According to our findings, H₂ concentrations in the species ranged from 3.6 to 48.9 μmol mg⁻¹ Chl a h⁻¹. H₂ production by isolated species was shown to have a 2% positive influence on oxygen (O₂) and carbon dioxide (CO₂) concentrations and a 2% negative effect on all nitrogen gas (N₂) concentrations. It was discovered that at high CO₂ concentrations, photosynthesis is enhanced but H₂ production is suppressed. Anabaena variabilis BTA-1047 was found to be the most active H₂-producing species, with an H₂ production activity of 21.3 μmol mg⁻¹ Chl a h⁻¹. Moreover, a 1% O₂: 2% CO₂ gas mixture doubled the strain activity of H₂ production. The findings of the study called into the question the notion that only an anaerobic environment is required for H₂ production by N₂-fixing cyanobacterial species and explored whether H₂ productivity can be increased by stimulating the micro-anaerobic environment with a carbon source.     

Reduction of tars by dolomite cracking during two-stage gasification of olive cake

The effective implementation of biomass gasification has to overcome some difficulties such as the minimization of tars. On the other hand, with a proper design of experimental conditions, biomass gasification can be directed towards the production of hydrogen. The aim of the present study was to investigate the use of dolomite as catalyst to improve tar removal and hydrogen production by a two-stage steam gasification process, using olive cake as raw material. Fixing the olive cake gasification conditions on the first reactor (900 °C, steam flow rate of 190 mg min⁻¹, O₂ flow rate of 7.5 cm³ min⁻¹), the cracking of tars was prompted by: a) steam gasification (steam flow rate in the range 40-190 mg min⁻¹) at 1000 °C, b) catalytic gasification, using dolomite (5% wt.). It was found that increasing steam flow rate up to 110 mg min⁻¹ involves an increase in hydrogen fraction due to the enhancement of water gas and water gas shift reactions. Also, the influence of dolomite was studied at 800 and 900 °C in a second reactor, finding better results at 800 °C, which gave an hydrogen fraction of 0.51.     

Biomass production and soil nutrients in organic and inorganic fertilized willow biomass production systems

The use of organic waste materials as nutrient sources for willow biomass production is an attractive means to decrease fertilization costs, increase biomass production and reduce greenhouse gas emissions associated with the system. In this study, changes in soil nutrients and biomass production of two willow varieties (Salix miyabeana–SX64 and Salix purpurea–9882-34) in organic and synthetic fertilized systems were compared at three locations in Northeastern U.S.A: Middlebury VT (MID), Delhi NY (DEL) and Fredonia NY (FRE). A 150 and 200 kg available N ha⁻¹ of urea as commercial fertilizer (CF), biosolid compost (BC) and digested dairy manure (DM) and a control (CT0) treatments were applied in June 2008 to the willow which was re-sprouting after coppice. There was no significant difference (p > 0.05) in biomass production among the fertilization treatments at any of the three sites and for either of the varieties. First rotation biomass production of 9882-34 ranged from 9.0 to 11.6 Mg ha⁻¹ yr⁻¹ at DEL, 3.4–8.8 Mg ha⁻¹ yr⁻¹ at MID and 3.5–7.7 Mg ha⁻¹ yr⁻¹ at FRE. For SX64, biomass production ranged from 13.2 to 19.0 Mg ha⁻¹ yr⁻¹ at DEL, 9.0–15.0 Mg ha⁻¹ yr⁻¹ at Mid and 5.5–9.3 Mg ha⁻¹ yr⁻¹ at FRE. SX64 deployed small numbers of large stems and produced more biomass than 9882-34 which deployed large numbers of small stems. Application of BC significantly increased soil N and P levels at MID in both 2008 and 2009 (p < 0.05). At DEL, BC and DM treatments increased soil N, Ca, Mg and OM levels in both 2008 and 2009 (p < 0.05). The fertilization treatments had no significant effect on any soil nutrients at FRE. This study indicates that willow biomass can be produced without fertilizer additions in the first rotation across this range of sites due to the nutrient status of these sites and high internal nutrient cycling in these systems.     

Effects of delayed winter harvest on biomass yield and quality of napiergrass and energycane

Napiergrass (Cenchrus purpureus (Schumach.) Morrone) and energycane (Saccharum hyb.) are perennial grasses that are well-suited for biomass production in the southeastern USA. The purpose of this study was to determine the effects of delayed winter harvest on biomass yield and quality of these grasses. The study was conducted on two adjacent sites near Midville, GA. Each site used a split-plot design with four replications, with species as the main plot, and harvest times (December, January, or February) as sub-plots. Dry matter (DM) yields were measured by mechanical harvesting, and a sample of biomass was taken from each harvest for determination of ethanol production by simultaneous saccharification and fermentation (SSF). Biomass moisture, N, P, K, and ash mass fractions were also measured. Energycane DM yields were stable from December (46.8 Mg ha⁻¹) to January (42.9 Mg ha⁻¹), but then declined (36.8 Mg ha⁻¹), while napiergrass yields declined sharply from December (47.0 Mg ha⁻¹) to January (35.0 Mg ha⁻¹). Napiergrass moisture mass fraction was reduced by an average of 18% in February harvests compared to December. Mass fractions of N, K, and ash tended to decrease with later harvesting, but sometimes increased due to changes in biomass composition. Delaying harvest of napiergrass from December to January reduced N removal by an average of 144 kg ha⁻¹, while delaying harvest of energycane to February reduced N removal by an average of 54 kg ha⁻¹. In SSF, later-harvested energycane produced less ethanol per unit of DM while napiergrass was less affected by harvest date.     

Bioconversion of crude glycerol from waste cooking oils into hydrogen by sub-tropical mixed and pure cultures

This study compared the biohydrogen generation by sub-tropical mixed and pure cultures from the crude glycerol from the biodiesel production using waste cooking oils (WCO). The crude glycerol was pretreated by pH adjustment. The mixed culture was obtained from a subtropical granular sludge of the UASB (Upflow Anaerobic Sludge Blanket) reactor used in the treatment of vinasse from sugarcane of ethanol and sugar industry. It was heat treated in order to inactivate hydrogen-consuming bacteria, which was identified by Illumina MiSeq Sequencing with a relative abundance of 97.96% Firmicutes Philum, 91.81% Clostridia Class and 91.81% Clostridiales Order. The pure culture was isolated from a sub-tropical granular sludge from UASB reactor of treating brewery wastewater and identified as Enterobacter sp. (KP893397). Two assays were carried in anaerobic batch reactors in order to verify the hydrogen production from crude glycerol bioconversion with: (I) mixed culture and (II) pure culture. The experiments were conducted at 37 °C, initial pH of 5.5 for assay I and 7.0 for assay II, with 20 g COD L⁻¹ of crude glycerol. The crude glycerol consumption was 56.2% and 88.0% for the assay I and II, respectively. The hydrogen yields were 0.80 moL H₂ mol⁻¹ glycerol for the assay I and 0.13 moL H₂ mol⁻¹ glycerol for the assay II. Enterobacter sp. preferred the reductive metabolic route, generating 1460.0 mg L⁻¹ of 1,3-propanediol, and it showed to be more sensitive in the presence of methanol from crude glycerol than mixed culture that preferred the oxidative metabolic route with biohydrogen generation. The mixed culture was more able to generate H₂ than pure culture from the crude glycerol coming from the biodiesel production using WCO.     

Biohydrogen production by Anabaena sp. PCC 7120 wild-type and mutants under different conditions: Light, nickel, propane, carbon dioxide and nitrogen

Increasing awareness of environmental problems caused by the current use of fossil fuel-based energy, has led to the search for alternatives. Hydrogen is a good alternative and the cyanobacterium Anabaena sp. PCC 7120 is naturally able to produce molecular hydrogen, photosynthetically from water and light. However, this H₂ is rapidly consumed by the uptake hydrogenase.This study evaluated the hydrogen production of Anabaena sp. PCC 7120 wild-type and mutants: hupL⁻ (deficient in the uptake hydrogenase), hoxH⁻ (deficient in the bidirectional hydrogenase) and hupL⁻/hoxH⁻ (deficient in both hydrogenases) on several experimental conditions, such as gas atmosphere (argon and propane with or without N₂ and/or CO₂ addition), light intensity (54 and 152 ??Em⁻²s⁻¹), light regime (continuous and light/dark cycles 16 h/8 h) and nickel concentrations in the culture medium.In every assay, the hupL⁻ and hupL⁻/hoxH⁻ mutants stood out over wild-type cells and the hoxH⁻ mutant. Nevertheless, the hupL⁻ mutant showed the best hydrogen production except in an argon atmosphere under 16 h light/8 h dark cycles at 54 ??Em⁻²s⁻¹ in the light period, with 1 ??M of NiCl₂ supplementation in the culture medium, and under a propane atmosphere.In all strains, higher light intensity leads to higher hydrogen production and if there is a daily 1% of CO₂ addition in the gas atmosphere, hydrogen production could increase 5.8 times, related to the great increase in heterocysts differentiation (5 times more, approximately), whereas nickel supplementation in the culture medium was not shown to increase hydrogen production. The daily incorporation of 1% of CO₂ plus 1% of N₂ did not affect positively hydrogen production rate.