Isolation and evaluation of a high H2-producing lab isolate from cow dung

Hydrogen producing bacterial strain was isolated from Indian cow dung and identified of the bacterial family Enterobacteriaceae. This lab isolate was differentiated from Citrobacter Y-19 at molecular level by using RAPD, PCR based technique, and OPO-03₄₆₀ and OPO-17₈₀₀ RAPD marker for this specific strain (lab isolate) was identified. Fermentative studies were investigated for important parameters, starting with pH of the culture, temperature, inoculum age and inoculum volume, initial substrate concentration and different substrates. Among different substrates, dextrose and sucrose were the preferred substrates for hydrogen production. The optimal starting pH of the culture was found to be 5.0. The H₂ production increased with increase in temperature up to 30 °C. The maximum value of H₂ production was recorded when inoculum volume was 12.5% of the culture broth and inoculum age was 14 h. Under batch fermentation conditions, the maximum hydrogen production rate and yield were 355.2 ml l⁻¹ h⁻¹ and 2.1 mol/mol glucose (conversion 35%), respectively. These results indicate that this lab isolate is an ideal hydrogen producer.     

Hydrogen–methane production from swine manure: Effect of pretreatment and VFAs accumulation on gas yield

A two-phase anaerobic process to produce hydrogen and methane from swine manure was investigated, using pretreated sludge with heat, acid and alkali treatment as inoculum. The relative order of pretreatment methods of H₂ productivity effectiveness and CH₄ productivity effectiveness produced by the residua of the first phase was heat treatment > alkali treatment > acid treatment. When the inoculum sludge was heat-treated at 80°C for 30 min, the H₂ and CH₄ production rate was the highest of 36.6, 201.7 ml (g TS)_added⁻¹. There were significant correlations between biogas production and accumulation of acetic acid and butyric acids. When propionic acid and total VFA concentrations reached about 2850 mg L⁻¹ and 10.0 g L⁻¹, respectively, the average H₂ production rate and H₂ content decreased from 7.6 ml d⁻¹(g VS)_added⁻¹ and 55.3% to 1.4 ml d⁻¹(g VS)_added⁻¹ and 43.2%, respectively. The activity of methanogenic bacteria was inhibited to a significant extent when the total VFA concentration was above 10.0 g L⁻¹, but this inhibitory effect weakened when the VFA concentration fell to 6200–8500 mg L⁻¹. Correspondingly, average CH₄ production rate increased from 4.0 ml d⁻¹(g TS)_added⁻¹ to 12.5 ml d⁻¹(g TS)_added⁻¹. Propionic acid was degraded rapidly only when acetic and butyric acid concentrations dropped to 2500 mg L⁻¹ and 1000 mg L⁻¹, respectively.     

Efficient hydrogen gas production from cassava and food waste by a two-step process of dark fermentation and photo-fermentation

A two-step process of sequential anaerobic (dark) and photo-heterotrophic fermentation was employed to produce hydrogen from cassava and food waste. In dark fermentation, the average yield of hydrogen was approximately 199 ml H₂ g⁻¹ cassava and 220 ml H₂ g⁻¹ food waste. In subsequent photo-fermentation, the average yield of hydrogen from the effluent of dark fermentation was approximately 611 ml H₂ g⁻¹ cassava and 451 ml H₂ g⁻¹ food waste. The total hydrogen yield in the two-step process was estimated as 810 ml H₂ g⁻¹ cassava and 671 ml H₂ g⁻¹ food waste. Meanwhile, the COD decreased greatly with a removal efficiency of 84.3% in cassava batch and 80.2% in food waste batch. These results demonstrate that cassava and food waste could be ideal substrates for bio-hydrogen production. And a two-step process combining dark fermentation and photo-fermentation was highly improving both bio-hydrogen production and removal of substrates and fatty acids.     

Optimization and microbial community analysis for production of biohydrogen from palm oil mill effluent by thermophilic fermentative process

The optimum values of hydraulic retention time (HRT) and organic loading rate (OLR) of an anaerobic sequencing batch reactor (ASBR) for biohydrogen production from palm oil mill effluent (POME) under thermophilic conditions (60 °C) were investigated in order to achieve the maximum process stability. Microbial community structure dynamics in the ASBR was studied by denaturing gradient gel electrophoresis (DGGE) aiming at improved insight into the hydrogen fermentation microorganisms. The optimum values of 2-d HRT with an OLR of 60 gCOD l⁻¹ d⁻¹ gave a maximum hydrogen yield of 0.27 l H₂ g COD⁻¹ with a volumetric hydrogen production rate of 9.1 l H₂ l⁻¹ d⁻¹ (16.9 mmol l⁻¹ h⁻¹). The hydrogen content, total carbohydrate consumption, COD (chemical oxygen demand) removal and suspended solids removal were 55 ± 3.5%, 92 ± 3%, 57 ± 2.5% and 78 ± 2%, respectively. Acetic acid and butyric acid were the major soluble end-products. The microbial community structure was strongly dependent on the HRT and OLR. DGGE profiling illustrated that Thermoanaerobacterium spp., such as Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium bryantii, were dominant and probably played an important role in hydrogen production under the optimum conditions. The shift in the microbial community from a dominance of T. thermosaccharolyticum to a community where also Caloramator proteoclasticus constituted a major component occurred at suboptimal HRT (1 d) and OLR (80 gCOD l⁻¹ d⁻¹) conditions. The results showed that the hydrogen production performance was closely correlated with the bacterial community structure. This is the first report of a successful ASBR operation achieving a high hydrogen production rate from real wastewater (POME).     

One-stage H2 and CH4 and two-stage H2 + CH4 production from grass silage and from solid and liquid fractions of NaOH pre-treated grass silage

In the present study, mesophilic CH₄ production from grass silage in a one-stage process was compared with the combined thermophilic H₂ and mesophilic CH₄ production in a two-stage process. In addition, solid and liquid fractions separated from NaOH pre-treated grass silage were also used as substrates. Results showed that higher CH₄ yield was obtained from grass silage in a two-stage process (467 ml g⁻¹ volatile solids (VS)_original) compared with a one-stage process (431 ml g⁻¹ VS_original). Similarly, CH₄ yield from solid fraction increased from 252 to 413 ml g⁻¹ VS_original whereas CH₄ yield from liquid fraction decreased from 82 to 60 ml g⁻¹ VS_original in a two-stage compared to a one-stage process. NaOH pre-treatment increased combined H₂ yield by 15% (from 5.54 to 6.46 ml g⁻¹ VS_original). In contrast, NaOH pre-treatment decreased the combined CH₄ yield by 23%. Compared to the energy value of CH₄ yield obtained, the energy value of H₂ yield remained low. According to this study, highest CH₄ yield (495 ml g⁻¹ VS_original) could be obtained, if grass silage was first pre-treated with NaOH, and the separated solid fraction was digested in a two-stage (thermophilic H₂ and mesophilic CH₄) process while the liquid fraction could be treated directly in a one-stage CH₄ process.     

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).     

Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16

Lignocellulosic biomass, if properly hydrolyzed, can be an ideal feedstock for fermentative hydrogen production. This work considered the pretreatment of corn stover (CS) using a dilute acid hydrolysis process and studied its fermentability for hydrogen production by the strain Thermoanaerobacterium thermosaccharolyticum W16. The effects of sulfuric acid concentration and reaction time in the hydrolysis stage of the process were determined based on a 2² central composite experimental design with respect to maximum hydrogen productivity. The optimal hydrolysis conditions to yield the maximum quantity of hydrogen by W16 were 1.69% sulfuric acid and 117 min reaction time. At these conditions, the hydrogen yield was shown to be 3305 ml H₂ L⁻¹ medium, which corresponds to 2.24 mol H₂ mol⁻¹ sugar. The present results indicate the potential of using T. thermosaccharolyticum W16 for high-yield conversion of CS hemicellulose into bio-H₂ integrated with acid hydrolysis.     

Photoproduction of H2 by wildtype Anabaena PCC 7120 and a hydrogen uptake deficient mutant: from laboratory experiments to outdoor culture

We have analyzed the filamentous cyanobacterium Anabaena PCC 7120 (wildtype) containing one nitrogenase, one uptake hydrogenase and one bidirectional hydrogenase and its hydrogen uptake deficient mutant AMC 414 for their H₂ production capacities. Anabaena PCC 7120 and AMC 414 had similar growth rates in turbidostat mode with increased growth rates at higher light intensity. Rates of C₂H₂ reduction were similar for both strains. In contrast to the wildtype, AMC 414 produced H₂ in a PhotoBioReactor (PhBR) using air as the lifting gas. The rate of H₂ production increased with light intensity and was not even saturated at 456 μEm⁻² s⁻¹. H₂ production increased significantly when replacing the air with argon. The maximal H₂ production during outdoor conditions was recorded using AMC 414 with a peak at 14.9 ml H₂ h⁻¹ l⁻¹. Despite the relatively high production, maximal efficiency of solar energy to H₂ conversion was only 0.042%. A molecular method was developed to analyze the relative abundancies of weight and mutant in competition experiments in the PhBR.     

Fed-batch operation for bio-H2 production by Rhodopseudomonas palustris (strain 42OL)

Interest in renewable and clean energies such as hydrogen has increased because of the high level of polluting emissions, increasing costs associated with petroleum and the escalating problems of global climate change. In the presence of a light source, a microbial photosynthetic process provides a system for the conversion of some organic compounds into biomass and hydrogen. Using Rhodopseudomonas palustris as a cell-factory, hydrogen photo-evolution was investigated in a photobioreactor (PBR) irradiated either from one or two opposite sides. Irradiating the photobioreactor from only one side, in the presence of malic acid, a reactor hydrogen production of 2.786 l(H₂) PBR⁻¹ was achieved. When the PBR was irradiated from two opposite sides, hydrogen photo-evolution increased to 3.162 l(H₂) PBR⁻¹. Experiments were carried out using inoculum from either the retardation or the exponential growth phases. Using the latter, the highest hydrogen photo-evolution rate based on the bacteriochlorophyll (Bchl) concentration was achieved (3295 μl(H₂) mg (Bchl⁻¹ h⁻¹). The hydrogen to biomass ratio (r_g) was 1.91 l g⁻¹ in the medium containing malic acid and 1.07 l g⁻¹ in that containing acetic acid. It was found that the hydrogen production rate was higher with malic than with acetic acid. Although photobiological hydrogen production cannot furnish alone the greater and greater world requirements of clean renewable energy, it is desirable that photobiological hydrogen technology will grow, in the near future, because photobioreactors for bio-hydrogen production can be positioned in fringe areas without competition with agricultural lands.     

Strategy for enhancing photo-hydrogen production yield by repeated fed-batch cultures

In order to obtain the high H₂ yield, photo-hydrogen production by Rhodopseudomonas faecalis strain RLD-53 in fed-batch culture using acetate as the sole carbon was studied. In repeated fed-batch culture, biomass increased rapidly from 0.05 to 0.65 g/l within 120 h, with the specific maximal growth rate estimated 0.68 × 10⁻³ g/h. After 120 h, biomass increased slowly and after each time feeding biomass increased slightly. The specific cumulative H₂ volumes in each phase were 2791.3, 1161.7, 1445.5 and 840.6 ml H₂/l-culture, respectively. The average H₂ yield was 3.17 mol H₂/mol acetate based on the whole process while the average substrate conversion efficiency reached 79.3%. Specific maximum H₂ production rate and H₂ content was 37.2 ml H₂/l/h and 95.5%, respectively. The results demonstrated the repeated fed-batch mode obtained higher efficiency for hydrogen production, feeding acetate concentration and control of pH were important to fed-batch culture hydrogen production.     

Hydrogen gas production from vinegar fermentation wastewater by electro-hydrolysis: Effects of initial COD content

Vinegar fermentation wastewater with different initial COD contents (9.66–48.6 g L⁻¹) were used for hydrogen gas production with simultaneous COD removal by electro-hydrolysis. The applied DC voltage was constant at 4 V. The highest cumulative hydrogen production (3197 ml), hydrogen yield (2766 ml H₂ g⁻¹ COD), hydrogen formation rate (799 ml d⁻¹), and percent hydrogen (99.5%) in the gas phase were obtained with the highest initial COD of 48.6 g COD L⁻¹. The highest energy efficiency (48%) was obtained with the lowest COD content of 9.66 g L⁻¹. Hydrogen gas production by water electrolysis was less than 250 ml and wastewater control resulted in less than 25 ml H₂ in 96 h. The highest (12%) percent COD removal was obtained with the lowest COD content. Hydrogen gas was produced by reaction of (H⁺) ions present in raw WW ( pH = 3.0) and protons released from acetic acid with electrons provided by electrical current. Electro-hydrolysis of vinegar wastewater was proven to be an effective method of H₂ gas production with some COD removal.     

Dark fermentative bio-hydrogen production from waste wheat starch using co-culture with periodic feeding: Effects of substrate loading rate

Dark fermentation experiments were performed for bio-hydrogen production from ground wheat starch solution (10 ± 1 g l⁻¹) using periodic feeding and effluent removal. A mixed culture of Clostridium butyricum-NRRL 1024 and Clostridium pasteurianum-NRRL B-598 were used with an initial biomass ratio of 1/1.Effects of wheat starch loading rate on the rate and yield of bio-hydrogen formation were investigated. Substrate loading rate was varied between 0.54 and 5.52 g d⁻¹ (HRT = 6-60 h). The highest hydrogen formation rate (280 ml d⁻¹), volumetric hydrogen formation rate (1857 ml H₂ l⁻¹ d⁻¹) and volatile fatty acids (VFAs) concentration were obtained with a substrate loading rate of 5.52 g d⁻¹ (HRT = 6 h). The highest hydrogen yield (109 ml H₂ g TS ⁻¹) was obtained with a substrate loading rate of 1.38 g d⁻¹.     

Biological hydrogen production from phenol-containing wastewater using Clostridium butyricum

Toxicity renders certain industrial effluents unfit for recovering its bioenergy content. An enriched single strain, Clostridium butyricum, was herein applied to fermentatively produce hydrogen from glucose in the presence of 200–1500 mg L⁻¹ of phenol. The enriched C. butyricum yielded hydrogen at approximately 1.4 mol H₂ mol⁻¹ glucose in the presence of 200–400 mg L⁻¹ phenol. Significant inhibition of cell metabolism was noted at phenol concentration >1000 mg L⁻¹. During glucose fermentation, phenol dosed at 200–400 mg L⁻¹ was partly co-degraded. Ethanol and acetate were the primary metabolites, whose yields increased with increasing phenol concentration. The present results revealed the potential to harvest hydrogen from a toxic (phenol-containing) wastewater.     

Effects of starch loading rate on performance of combined fed-batch fermentation of ground wheat for bio-hydrogen production

Ground wheat powder solution (10 g L⁻¹) was subjected to combined dark and light fermentations for bio-hydrogen production by fed-batch operation. A mixture of heat treated anaerobic sludge (AN) and Rhodobacter sphaeroides-NRRL (RS-NRRL) were used as the mixed culture of dark and light fermentation bacteria with an initial dark/light biomass ratio of 1/2. Effects of wheat starch loading rate on the rate and yield of bio-hydrogen formation were investigated. The highest cumulative hydrogen formation (CHF = 3460 ml), hydrogen yield (201 ml H₂ g⁻¹ starch) and formation rate (18.1 ml h⁻¹) were obtained with a starch loading rate of 80.4 mg S h⁻¹. Complete starch hydrolysis and glucose fermentation were achieved within 96 h of fed-batch operation producing volatile fatty acids (VFA) and H₂. Fermentation of VFAs by photo-fermentation for bio-hydrogen production was most effective at starch loading rate of 80.4 mg S h⁻¹. Hydrogen formation by combined fermentation took place by a fast dark fermentation followed by a rather slow light fermentation after a lag period.     

Bio-methanization of energy crops through mono-digestion for continuous production of renewable biogas

The aim of this laboratory-scale study was to investigate the long-term anaerobic fermentation of an extremely sour substrate, an energy crop, for continuous production of methane (CH₄) as a source of renewable energy. The sugar beet silage was used as the mono-substrate, which had a low pH of around 3.3–3.4, without the addition of manure. The mesophilic biogas digester was operated in a hydraulic retention time (HRT) range between 15 and 9.5 days, and an organic loading rate (OLR) range of between 6.33 and 10 g VS l⁻¹ d⁻¹. The highest specific gas production rate (spec. GPR) and CH₄ content were 0.67 l g VS⁻¹ d⁻¹ and 74%, respectively, obtained at an HRT of 9.5 days and OLR of 6.35 g VS l⁻¹ d⁻¹. The digester worked within the neutral pH range as well. Since this substrate lacked the availability of macro and micro nutrients, and the buffering capacity as well, external supplementation was definitely required to provide a stable and efficient operation, as provided using NH₄Cl and KHCO₃ in this case. The findings of this ongoing long-term fermentation of an extremely acidic biomass substrate without manure addition have reflected crucial information about how to appropriately maintain the operational and particularly the environmental parameters in an agricultural biogas plant.     

The photoinduced evolution of O2 and H2 from a WO3 aqueous suspension in the presence of Ce/Ce

This is a report on the production of O₂ and H₂ from photocatalytic and photochemical processes in the WO₃–H₂O–Ce⁴⁺_aq–hν system. The photoproduction of O₂ and H₂ was studied over the range of WO₃ concentrations from 2 to 8 g dm⁻³, and conduction band electron scavenger concentrations 1–20 mM Ce_aq⁴⁺. Medium and high concentrations of the electron scavenger gave mainly O₂ as the main product. Dilute solutions of [Ce_aq⁴⁺]< 2 mM initially produced dioxygen, and then hydrogen after an induction period of 3–4 h. Yields of 140–250 μmol O₂  h⁻¹ and 1–7 μmol H₂ h⁻¹ were obtained and were found to depend on the physical properties and content of WO₃, the concentration of the electron scavenger, illumination period and wavelength, and the radiation geometry. The photoactivity of the suspension was correlated to the level of crystallinity of WO₃ powders. The studied system utilizes WO₃ to accomplish the initial light absorption, charge separation, and production of O₂ and H⁺ from the interaction of water molecules with photogenerated WO₃ valence band holes, in the presence of Ce⁴⁺_aq species as a scavenger of conduction band electrons. This is followed by the evolution of H₂ from a homogeneous photochemical reduction of H⁺ and/or H₂O by photoexcited Ce³⁺_aq, formed from the earlier reduction of Ce⁴⁺_aq. The obtained results show that, with an appropriate design, tungsten trioxide is a promising material that can be used as a photoactive component in energy conversion systems or in environmental photocatalysis, using artificial or solar light.     

Repeated production of hydrogen by sulfate re-addition in sulfur deprived culture of Chlamydomonas reinhardtii

Biological hydrogen production by the green alga, Chlamydomonas reinhardtii can be induced in conditions of sulfur deprivation. In this study, we investigated the repeated and enhanced hydrogen production afforded by the re-addition of sulfate with monitoring of pH and concentration of chlorophyll and sulfate. Without adjustment of the pH, the optimal concentration of re-added sulfate was 30 μM for the hydrogen production. By the re-addition of 30 μM of sulfate and the adjustment of the pH during 4 cycles of repeated production, we obtained the maximum amount of 789 ml H₂ l⁻¹ culture, which is 3.4 times higher than that of one batch production without adjustment of pH, 236 ml H₂ l⁻¹ culture. This means that the enhancement of the hydrogen production can be achieved by the careful control of the sulfate re-addition and pH adjustment in the sulfur deprived culture.     

Biohydrogen production from beet molasses by sequential dark and photofermentation

Biological hydrogen production using renewable resources is a promising possibility to generate hydrogen in a sustainable way. In this study, a sequential dark and photofermentation has been employed for biohydrogen production using sugar beet molasses as a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup⁻ mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NH₄⁺ and yeast extract on sucrose consumption and hydrogen production was determined. The highest hydrogen yield (4.2 mol of H₂/mol sucrose) and maximum volumetric productivity (7.1 mmol H₂/L_c.h) were obtained in the absence of NH₄⁺. The effluent of the dark fermentation containing no NH₄⁺ was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, iron-citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H₂/L_c.h) were attained using the hup⁻ mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H₂/mol sucrose in dark fermentation to 13.7 mol H₂/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H₂/mole of sucrose) by sequential dark and photofermentation.     

Bio-hydrogen production from acid hydrolyzed wheat starch by photo-fermentation using different Rhodobacter sp

Hydrogen gas production from sugar solution derived from acid hydrolysis of ground wheat starch by photo-fermentation was investigated. Three different pure strains of Rhodobacter sphaeroides (RV, NRLL and DSZM) were used in batch experiments to select the most suitable strain. The ground wheat was hydrolyzed in acid solution at pH = 3 and 90 °C in an autoclave for 15 min. The resulting sugar solution was used for hydrogen production by photo-fermentation after neutralization and nutrient addition. R. sphaeroides RV resulted in the highest cumulative hydrogen gas formation (178 ml), hydrogen yield (1.23 mol H₂ mol⁻¹ glucose) and specific hydrogen production rate (46 ml H₂ g⁻¹ biomass h⁻¹) at 5 g l⁻¹ initial total sugar concentration among the other pure cultures. Effects of initial sugar concentration on photo-fermentation performance were investigated by varying sugar concentration between 2.2 and 13 g l⁻¹ using the pure culture of R. sphaeroides RV. Cumulative hydrogen volume increased from 30 to 232 ml when total sugar concentration was increased from 2.2 to 8.5 g l⁻¹. Further increases in initial sugar concentration resulted in decreases in cumulative hydrogen formation. The highest hydrogen formation rate (3.69 ml h⁻¹) and yield (1.23 mol H₂ mol⁻¹ glucose) were obtained at a sugar concentration of 5 g l⁻¹.     

Bio-hydrogen production from cornstalk wastes by orthogonal design method

One-factor-at-a-time design and orthogonal design were used in the experimental design methods to optimize bio-hydrogen (bio-H₂) production from cornstalk wastes by anaerobic fermentation. Three series of experiments were designed to investigate the effects of substrate concentration, initial pH and orthogonal design on the bio-H₂ production by using the natural sludge as inoculant. Experimental results indicate that substrate concentration was the most significant condition for optimal hydrogen production. The optimum orthogonal design method was proposed to be at an enzymatic temperature of 50 °C, an enzymatic time of 72 h, an initial pH of 7.0 and a substrate concentration of 10 g/L. The proposed method facilitated the optimization of optimum design parameters, only with a few well-defined experimental sets. Under the proposed condition, the maximum cumulative H₂ yield was 141.29 ml g^?1-CS (cornstalk, or 164.48 ml g^?1-TS, total solid, TS = 0.859 W_{dried cornstalk}), with an average H₂ production rate of 12.31 ml g^?1-CS h^?1. The hydrogen content reached 57.85% and methane was not detected in the biogas.