The growth of oleaginous Rhodotorula glutinis in an internal-loop airlift bioreactor by using mixture substrates of rice straw hydrolysate and crude glycerol

The conversion of lignocellulosic biomass (LCB) to microbial oils is attracting a growing amount of attention. However, the growth of the oleaginous yeast Rhodotorula glutinis on LCB hydrolysate (mainly rice straw) only will lead to a low lipid mass fraction, in the range of 10–20%. This study shows that the addition of crude glycerol to the LCB hydrolysate medium can efficiently raise the lipid mass fraction to the range of 30–40%. Crude glycerol is a by-product in the biodiesel production process. The use of renewable LCB hydrolysate and crude glycerol would greatly reduce the substrate cost for microbial oil production using R. glutinis. In addition, the results of experiments show that a low-cost airlift bioreactor is a more suitable fermentation process for the growth of R. glutinis than the use of a conventional agitation tank. When using mixed carbon sources of LCB hydrolysate with 30 kg m⁻³ of reducing sugars and 30 kg m⁻³ of crude glycerol, a maximal cell mass of 21.4 kg m⁻³ and lipid mass fraction of 58.5 ± 6.2 were achieved in an internal loop airlift bioreactor, and this process may have the potential to be applied in scale-up production.     

Co-utilization of glucose,xylose and cellobiose by the oleaginous yeast Cryptococcus curvatus

Simultaneous utilization of mixed sugars is one of the major challenges for biofuel production utilizing lignocellulosic biomass as feedstock. Our previous work proved that the oleaginous yeast Cryptococcus curvatus could efficiently produce lipids, the precursors of hydrocarbons with high energy density, from lignocellulosic hydrolysates. However, the strain's capability of simultaneously utilizing primary sugars was still unknown. Thus, this work comprehensively explored the co-utilization of glucose, xylose and cellobiose by C. curvatus. The results indicated that the consumption of both xylose and cellobiose was repressed by glucose, while xylose and cellobiose could be simultaneously consumed at similar rates. The total sugar consumption rates remained constant at about 0.6 g L⁻¹ h⁻¹ regardless of the sugar composition in the mixtures, and the cell biomass and lipid production by C. curvatus cultured on the different sugar mixtures were similar. Moreover, compared with glucose and xylose, cellobiose could lead to higher dry cell weights and lipid yields, suggesting an excellent carbon source for lipid production. Based on these findings, this study demonstrated an effective approach for alleviating glucose repression for microbial lipid production by C. curvatus through xylose/cellobiose co-utilization which would greatly contribute to a more efficient and economical cellulosic biofuel production.     

Fermentative hydrogen production by Clostridium butyricum CGS5 using carbohydrate-rich microalgal biomass as feedstock

In this work, a carbohydrate-rich microalga, Chlorella vulgaris ESP6, was grown photoautotrophically to fix the CO₂. The resulting microalgal biomass was hydrolyzed by acid or alkaline/enzymatic treatment and was then used for biohydrogen production with Clostridium butyricum CGS5. The C. vulgaris biomass could be effectively hydrolyzed by acid pretreatment while similar hydrolysis efficiency was achieved by combination of alkaline pretreatment and enzymatic hydrolysis. The biomass of C. vulgaris ESP6 containing a carbohydrate content of 57% (dry weight basis) was efficiently hydrolyzed by acid treatment with 1.5% HCl, giving a reducing sugars (RS) yield of nearly 100%. C. butyricum CGS5 could utilize RS from C. vulgaris ESP6 biomass to produce hydrogen without any additional organic carbon sources. The optimal conditions for hydrogen production were 37 °C and a microalgal hydrolysate loading of 9 g RS/L with pH-controlled at 5.5. Under the optimal conditions, the cumulative H₂ production, H₂ production rate, and H₂ yield were 1476 ml/L, 246 ml/L/h, and 1.15 mol/mol RS, respectively. The results demonstrate that the C. vulgaris biomass has the potential to serve as effective feedstock for dark fermentative H₂ production.     

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.     

Culture of microalgal strains isolated from natural habitats in Thailand in various enriched media

Six freshwater microalgal strains in the class of Chlorophyceae, including Chlorococcum humicola, Didymocystis bicellularis, Monoraphidium contortum, Oocystis parva, Sphaerocystis sp., and Scenedesmus acutus were isolated from natural habitats in Thailand. The six strains were compared for their biomass yield, lipid content, and lipid productivity in four enriched culture media in batch mode. Significant differences were found across algal strains and culture media. The best strain was found to be C. humicola, which had the highest biomass yield of 0.113 g/l/d (in Kuhl medium), the highest lipid content of 45.94% (in BG-11 medium), and the highest lipid yield of 0.033 g/l/d (in 3NBBM medium). The 3NBBM medium, which has the lowest nitrogen concentration among the four culture media, was considered the optimal culture medium for C. humicola for lipid production. The fatty acid profile of C. humicola was also found to be affected by the culture medium. More oleic acid (C18:1) but less linolenic acid (C18:3) was accumulated in BG-11 and 3NBBM than in Kuhl and N-8 media. Lipid profiles of C. humicola were comparable to palm oil in the percentage of palmitic acid and the total amount of saturated fatty acids; however, C. humicola made more poly-unsaturated fatty acids such as linoleic (C18:2) and linolenic (C18:3) acids than oil palms. Lipids from C. humicola were believed to be acceptable for biodiesel production.     

Single-stage photo-fermentative hydrogen production from hydrolyzed straw biomass using Rhodobacter sphaeroides

The hydrogen production utilizing photosynthetic and anaerobic bacteria in two-stage approach has many drawbacks, such as shortage of raw materials and complexity of operations. Accordingly, we aimed to develop a simple one-stage H₂ production protocol using the depolymerization of maize straw cellulose as a cheap carbon source. R. sphaeroides HY01 and its mutant (Hup^?) were studied regarding their H₂ production under different culture conditions. Further study using two model sugars, their combination, and straw hydrolysate as carbon sources was conducted to determine the effects of substrate on H₂ production. When using the straw hydrolysate as carbon source, the pH remained in a range of 7.1–7.6, whereas it dropped to 5.4–7.4 when using the model sugars, and the former biomass value was greater. The H₂ production performance of the mutant was significantly better than that of HY01. One-step photo-fermentative H₂ production was superior when using straw hydrolysate as opposed to the simple model sugars, and its yield was up to 4.62 mol H₂·mol^?1 reducing sugar.     

Production of biohydrogen from sugars and lignocellulosic biomass using Thermoanaerobacter GHL15

The effect of culture parameters on hydrogen production using strain GHL₁₅ in batch culture was investigated. The strain belongs to the genus Thermoanaerobacter with 98.9% similarity to Thermoanaerobacter yonseiensis and 98.5% to Thermoanaerobacter keratinophilus with a temperature optimum of 65–70 °C and a pH optimum of 6–7. The strain metabolizes various pentoses, hexoses, and disaccharides to acetate, ethanol, hydrogen, and carbon dioxide. However substrate inhibition was observed above 10 mM glucose concentration. Maximum hydrogen yields on glucose were 3.1 mol H₂ mol⁻¹ glucose at very low partial pressure of hydrogen. Hydrogen production from various lignocellulosic biomass hydrolysates was investigated in batch culture. Various pretreatment methods were examined including acid, base, and enzymatic (Celluclast^® and Novozyme 188) hydrolysis. Maximum hydrogen production (5.8–6.0 mmol H₂ g⁻¹ dw) was observed from Whatman paper (cellulose) hydrolysates although less hydrogen was produced by hydrolysates from other examined lignocellulosic materials (maximally 4.83 mmol H₂ g⁻¹ dw of grass hydrolysate). The hydrogen yields from all lignocellulosic hydrolysates were improved by acid and alkaline pretreatments, with maximum yields on grass, 7.6 mmol H₂ g⁻¹ dw.     

Photofermentive production of biohydrogen from oil palm waste hydrolysate

Oil palm empty fruit bunch (OPEFB) was hydrolyzed with dilute sulfuric acid (6% v/v; 8 mL acid per g dry OPEFB) at 120 °C for 15-min to release the fermentable sugars. The hydrolysate contained xylose (23.51 g/L), acetic acid (2.44 g/L) and glucose (1.80 g/L) as the major carbon components. This hydrolysate was used as the sole carbon source for photofermentive production of hydrogen using a newly identified photosynthetic bacterium Rhodobacter sphaeroides S10. A Plackett–Burman experimental design was used to examine the influence of the following on hydrogen production: yeast extract concentration, molybdenum concentration, magnesium concentration, EDTA concentration and iron concentration. These factors influenced hydrogen production in the following decreasing order: yeast extract concentration > molybdenum concentration > magnesium concentration > EDTA concentration > iron concentration. Under the conditions used (35 °C, 14.6 W/m² illumination, initial pH of 7.0), the optimal composition of the culture medium was (per L): mixed carbon in OPEFB hydrolysate 3.87 g, K₂HPO₄ 0.9 g, KH₂PO₄ 0.6 g, CaCl₂⋅2H₂O 75 mg, l-glutamic acid 795.6 mg, FeSO₄⋅7H₂O 11 mg, Na₂MoO₂⋅2H₂O 1.45 mg, MgSO₄⋅7H₂O 2.46 g, EDTA 0.02 g, yeast extract 0.3 g). With this medium, the lag period of hydrogen production was 7.65 h, the volumetric production rate was 22.4 mL H₂/L medium per hour and the specific hydrogen production rate was 7.0 mL H₂/g (xylose + glucose + acetic acid) per hour during a 90 h batch culture of the bacterium. Under optimal conditions the conversion efficiency of the mixed carbon substrate to hydrogen was nearly 29%.     

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

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

Biohydrogen and 5-aminolevulinic acid production from waste barley by Rhodobacter sphaeroides O.U.001 in a biorefinery concept

The production of biohydrogen and 5-aminolevulinic acid (5-ALA) by Rhodobacter sphaeroides O.U.001 was investigated in a biorefinery concept. Waste barley was used as a substrate after acid hydrolysis. The hydrolysate was analyzed in terms of its total simple sugar, organic acid, ammonium, element and total phenol contents. Four different growth media having 5 g/L, 7 g/L, 9 g/L and 11 g/L sugar content were prepared using the waste barley hydrolysate to produce biohydrogen and 5-ALA. The increased sugar concentrations resulted in higher cell density and hydrogen accumulation. Accordingly, the highest cell density (OD₆₆₀: 1.78) and hydrogen production (0.4 L H₂/L culture) were observed in the 11 g/L sugar-containing medium. A 67.4 μM 5-ALA was produced upon vitamin B₁₂ and levulinic acid additions. These results showed that waste barley can be used as a substrate for R. sphaeroides for biohydrogen and 5-ALA production within a biorefinery concept.     

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

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

Detoxification of woody hydrolyzates with activated carbon for bioconversion to ethanol by the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum

Autohydrolysis is a simple, green method of recovering sugars from biomass, using only hot water. One potential drawback is that byproducts are formed during the autohydrolysis process that could interfere with subsequent hydrolysis and fermentation to ethanol. In the present work, autohydrolysis prehydrolyzate from mixed hardwood chips was detoxified with activated carbon and the removal efficiency of byproducts as well as the loss of sugars determined. The resulting detoxified prehydrolyzate was evaluated for the fermentation to ethanol with a thermophilic anaerobic bacterium. Activated carbon at a 2.5 wt % level on the prehydrolyzate was able to remove 42% of formic acid, 14% of acetic acid, 96% of hydroxymethylfurfural (HMF) and 93% of the furfural. However, 8.9% of sugars were also removed. The removal of HMF and furfural follow expected adsorption isotherms but formic acid, acetic acid, and sugars did not. Autohydrolysis prehydrolyzates from mixed hardwood detoxified with activated carbon can be fermented with Thermoanaerobacterium saccharolyticum strain MO1442 with an essentially 100% yield. T. saccharolyticum strain MO1442 is able to metabolize the glucose, xylose, and arabinose in the hydrolyzate. The results showed the detoxification process with activated carbon improved the ethanol yields by the removal of toxic compounds, mainly HMF and furfural, with moderate loss of fermentable sugars.     

Evaluating renewable carbon sources as substrates for single cell oil production by Cunninghamella echinulata and Mortierella isabellina

The biochemical behavior (biomass production, accumulation of total lipid, substrate uptake, fatty acid composition of fungal oil) of two oleaginous Mucorales strains, namely Mortierella isabellina ATHUM 2935 and Cunninghamella echinulata ATHUM 4411, was studied when the aforementioned microorganisms were cultivated on xylose, raw glycerol and glucose under nitrogen-limited conditions. Significant differences in the process of lipid accumulation as related to the carbon sources used were observed for both microorganisms. These differences were attributed to the different metabolic pathways involved in the assimilation of the above substrates. Therefore, the various carbon sources were channeled, at different extent, to storage lipid or to lipid-free biomass formation. Although glucose containing media favored the production of mycelial mass (15 g L^?1 of total biomass in the case of C. echinulata and 27 g L^?1 in the case of M. isabellina), the accumulated lipid in dry matter was 46.0% for C. echinulata and 44.6% for M. isabellina. Lipid accumulation was induced on xylose containing media (M. isabellina accumulated 65.5% and C. echinulata 57.7% of lipid, wt wt^?1, in dry mycelial mass). In these conditions, lipids of C. echinulata contained significant quantities of γ-linolenic acid (GLA). This fungus, when cultivated on xylose, produced 6.7 g L^?1 of single cell oil and 1119 mg L^?1 of GLA. Finally, the growth of both C. echinulata and M. isabellina on raw glycerol resulted in lower yields in terms of both biomass and oil produced than the growth on xylose.     

Biotechnological conversions of biodiesel derived waste glycerol by yeast and fungal species

Fifteen eukaryotic microorganisms were tested for their ability to assimilate biodiesel derived waste glycerol and convert it into value-added metabolic products. For this purpose yeast and Zygomycetes strains were cultivated in nitrogen-limited raw glycerol-based media (initial glycerol concentration 30 g/L). Yeasts tested accumulated restricted lipid quantities (up to ∼22%, wt/wt, in the case of Rhodotorula sp), while differentiations in their fatty acid composition were recorded in relation to the yeast strains employed and the fermentation time. On the contrary, fungi accumulated higher quantities of lipid inside their mycelia (ranging between 18.1 and 42.6%, wt/wt, of dry biomass) that contained in variable amounts the medically important GLA (γ-linolenic acid). Moreover, Yarrowia lipolytica, Pichia membranifaciens and Thamnidium elegans were further studied in media having increased initial glycerol concentrations. In these conditions Y. lipolytica secreted significant amounts of acetic acid (29.2 g/L), as well as mannitol (19.4 g/L) while P. membranifaciens reached 28.4 g/L of biomass at glycerol concentration 90 g/L. T. elegans produced 11.6 g/L of oil, with 71.1%, wt/wt, of fat in biomass, while the maximum concentration of GLA was 371 mg/L. Detailed analysis of T. elegans lipids indicated that the phospholipids fraction was particularly rich in polyunsaturated fatty acids.     

Photo-fermentative hydrogen production by Rhodopseudomonas palustris CGA009 in the presence of inhibitory compounds

This study investigated Rhodopseudomonas palustris CGA009 biohydrogen production from compounds commonly found in lignocellulosic steam explosion hydrolysate, by examining the effect of individual inhibitory phenolic and furan compounds found in hydrolysates, under photo-fermentative anaerobic conditions. Since lignocellulose is often converted into ethanol via yeast-mediated fermentation, the tolerance of R. palustris CGA009 towards ethanol inhibition was also tested at a concentration range of 0.25–14% (v/v) under anaerobic photo-fermentative conditions. Hydrogen production was enhanced by compounds such as syringaldehyde (0.03 g/L), which accumulated total hydrogen of 960 mL over the cultivation period. In contrast, a reduction in hydrogen production of 1.4 fold was observed in vanillin-containing solutions (0.43 g/L), which obtained accumulated total hydrogen of 576 mL. Increasing ethanol concentrations reduced hydrogen production, but cell growth was not affected up to 1% (v/v), a fairly low concentration. R. palustris CGA009 can tolerate comparatively high concentrations of phenolic compounds, suggesting its use for lignocellulose hydrolysate detoxification and hydrogen production.     

Evaluation of an adapted inhibitor-tolerant yeast strain for ethanol production from combined hydrolysate of softwood

In order to evaluate the potential of an adapted inhibitor-tolerant yeast strain developed in our lab to produce ethanol from softwood, the effect of furfural and HMF presented in defined medium and pretreatment hydrolysate on cell growth was investigated. And the efficiency of ethanol production from enzymatic hydrolysate mixed with pretreatment hydrolysate of softwood by bisulfite and sulfuric acid pretreatment process was reported. The results showed that in the combined treatments of the two inhibitors, cell growth was not affected at 1 g/L each of furfural and HMF. When 3 g/L each of furfural and HMF was applied, the adapted strain responded with an extended lag phase of 24 h. Both in batch and fed-batch runs of combined hydrolysate fermentation, the final ethanol concentrations were above 20.0 g/L and the ethanol yields (Y_p/s) on the total amount of fermentable sugar presented in the pretreated materials were above 0.40 g/g. It implies the great promise of the yeast strain for improving ethanol production from softwood due to its high ability of metabolizing inhibitor compounds of furfural and HMF.     

Hydrolysis of pretreated rice straw by an enzyme cocktail comprising acidic xylanase from Aspergillus sp. for bioethanol production

The most crucial enzyme involved in xylan hydrolysis is endoxylanase which cleaves the internal glycosidic bonds of xylan. The aim of this work was to study the production of extracellular xylanase by a locally isolated strain of Aspergillus sp. under solid-state fermentation (SSF) and to evaluate the potential of the enzyme in enzymatic hydrolysis of pretreated rice straw. Xylanase production reached maximum with incubation period (96 h), moisture level (80%), inoculum size (3 × 10⁶ spores/mL), pH (4.8), temperature (25 °C), carbon source (wheat bran) and nitrogen source (yeast extract). Under optimized conditions, xylanase production reached to 5059 IU/gds. Crude xylanase was used for supplementing the enzyme cocktail comprising cellulases (Zytex, India), β-glucosidase (In-house) and xylanase (In-house) for the saccharification of alkali-pretreated rice straw to get the maximum reducing sugar production. The cocktail containing the three enzymes resulted a maximum of 574.8 mg/g of total reducing sugars in comparison to 430.2 mg/g sugars by the cocktail without xylanase. These results proved that the crude xylanase preparation from Aspergillus sp. could be a potent candidate for the enzyme cocktail preparation for biomass hydrolysis in lignocellulosic bioethanol program.     

Presence of low concentrations of acetic acid improves yeast tolerance to hydroxymethylfurfural (HMF) and furfural

BackgroundHydrolysates derived from lignocellulosic material contain a complex mix of inhibitory compounds dependant on the type of biomass and the pre-treatment process employed. These inhibitors prevent the subsequent fermentation of available sugars by yeast into ethanol.ResultsInhibitory compounds normally work synergistically to reduce metabolic output, rates of budding and viability; however, it was observed in this study that the presence of weak acids actually improved tolerance to hydroxymethyl furfural (HMF) and furfural in Saccharomyces cerevisiae. The protective role of weak acids in HMF or furfural stressed cells was only apparent with relatively low concentrations of acetic acid (20 mM), however, there was an improvement in glucose utilisation and ethanol production when compared with HMF or furfural stressed cells. Focusing on HMF stressed cells quantitative trait loci (QTL) analysis identified a region on chromosome VI related to the enhanced tolerance to HMF in the presence of acetic acid. Two genes FET5 and HAC1 located in this region were up-regulated under the combined stress of acetic acid with HMF stress and null mutants exhibited a return to HMF sensitivity.ConclusionsPresence of acetic acid helps yeast cells overcome HMF stress, QTL analysis identified two genes on a loci on chromosome VI, knocking out these genes returns the cell to HMF sensitivity.     

The addition of hydrolyzed rice straw in xylose fermentation by Pichia stipitis to increase bioethanol production at the pilot-scale

The pretreatment of agricultural biomass by diluted acid is often employed to facilitate the release of monosaccharide for the subsequent enzyme hydrolysis for lignocellulosic ethanol production. However, furfural and hydroxymethylfurfural are usually generated and markedly decrease the yield of pentose fermentation during this pretreatment. In the present study, the enhancement of lignocellulosic ethanol production was successfully demonstrated at pilot scale with extra addition of hydrolyzed rice straw into pentose fermentation by Pichia stiptis. This way has resulted into the increase of P. stiptis cell mass was shown to play a positive role. The ethanol yield, 0.45 g_p/g_s, with the addition of hydrolyzed rice straw in hemicellulosic hydrolysate from plywood, bagasse and bamboo were increase 20–51% to demonstrate the applicability of this technology in a variety of lignocellulosic ethanol processes due to the efficient conversion of xylose.     

Enzymatic saccharification and fermentation of paper and pulp industry effluent for biohydrogen production

Paper and pulp industry effluent was enzymatically hydrolysed using crude cellulase enzyme (0.8–2.2FPU/ml) obtained from Trichoderma reesei and from the hydrolysate biohydrogen was produced using Enterobacter aerogenes. The influence of temperature and incubation time on enzyme production was studied. The optimum temperature for the growth of T. reesei was found to be around 29 °C. The enzyme activity of 2.5 FPU/ml was found to produce about 22 g/l of total sugars consisting mainly of glucose, xylose and arabinose. Relevant kinetic parameters with respect to sugars production were estimated using two fraction model. The enzymatic hydrolysate was used for the biohydrogen production using E. aerogenes. The growth data obtained for E. aerogenes were fitted well with Monod and Logistic equations. The maximum hydrogen yield of 2.03 mol H₂/mol sugar and specific hydrogen production rate of 225 mmol of H₂/g cell/h were obtained with an initial concentration of 22 g/l of total sugars. The colour and COD of effluent was also decreased significantly during the production of hydrogen. The results showed that the paper and pulp industry effluent can be used as a substrate for biohydrogen production.