Influence of substrate concentration on the stability and yield of continuous biohydrogen production

The effect of substrate concentration (sucrose) on the stability and yield of a continuous fermentative process producing hydrogen was studied. High substrate concentrations are attractive from an energy standpoint as they would minimise the energy required for heating. The reactor was a CSTR; temperature was maintained at 35 degrees C; pH was controlled between 5.2 and 5.3, and the hydraulic retention time (HRT) was 12 h. Online measurements were taken for ORP, pH, temperature, %CO2, gas output and %H2, and data logged using a MatLAB data acquisition toolbox. Steady-state operation was obtained at 10, 20 and 40 g/L of sucrose in the influent, but a subsequent step change to 50 g/L was unsustainable. The hydrogen content ranged between 50% and 60%. The yield of hydrogen decreased as the substrate concentration increased from 1.7 +/- 0.2 mol/mol hexose added at 10 g/L, to 0.8 +/- 0.1 mol/mol at 50 g/L. Sparging with nitrogen improved the hydrogen yield by at least 35% at 40 g/L and at least 33% at 50 g/L sucrose. Sparging also enabled steady-state operation at 50 g/L sucrose. Addition of an extra 4 g/L of n-butyric acid to the reactor operating at 40 g/L sucrose increased the butyrate concentration from 9,830 to 18,900 mg/L, immediately stopping gas production and initiating the production of propionate, whilst the addition of 2 g/L taking the butyrate concentration to 12,200 mg/L did not do so. It was shown that operation at 50 g/L sucrose in a CSTR in butyrate fermentation is possible.     

Fermentation of Cellulose to Methane and Carbon Dioxide by a Rumen Anaerobic Fungus in a Triculture with Methanobrevibacter sp. Strain RA1 and Methanosarcina barkeri

The fermentation of cellulose by a rumen anaerobic fungus in the presence of Methanobrevibacter sp. strain RA1 and Methanosarcina barkeri strain 227 resulted in the formation of 2 mol each of methane and carbon dioxide per mol of hexose fermented. Coculture of the fungus with either Methanobrevibacter sp. or M. barkeri produced 0.6 and 1.3 mol of methane per mol of hexose, respectively. Acetate, formate, ethanol, hydrogen, and lactate, which are major end products of cellulose fermentation by the fungus alone, were either absent or present in very low quantities at the end of the triculture fermentation (≤0.08 mol per mol of hexose fermented). During the time course of cellulose fermentation by the triculture, hydrogen was not detected (<1 × 10⁻⁵ atm; <0.001 kPa) and only acetate exhibited transitory accumulation; the maximum was equivalent to 1.4 mol per mol of hexose at 6 days which was higher than the total acetate yield of 0.73 in the fungus monoculture. The effect of methanogens is interpreted as a shift in the flow of electrons away from the formation of electron sink products lactate and ethanol to methane via hydrogen, favoring an increase in acetate, which is in turn converted to methane and carbon dioxide by M. barkeri. The maximum rate of cellulose degradation in the triculture (3 mg/ml per day) was faster than previously reported for bacterial cocultures and within 16 days degradation was complete. The triculture was used successfully also in the production of methane from cellulose in the plant fibrous materials, sisal (fiber from leaves of Agave sisalona L.) and barley straw leaf.     

Enhancement of methane production from long chain fatty acid based effluents

Two anaerobic sludges previously loaded with oleate and palmitate accumulated 4570+/-257 and 5200+/-9 mgCOD-LCFAgVSS(-1), respectively. These sludges were incubated in batch assays and methane production was recorded after addition of 100-900 mg L(-1) of oleate and palmitate, respectively. The batch assays were conducted before and after allowing the depletion of the biomass-associated LCFA. The presence of biomass-associated LCFA decreased the capacity of both sludges to convert the added LCFA to methane. After the degradation of biomass-associated LCFA, the lag phases observed before the onset of methane production were significantly reduced, evidencing an increase in the tolerance of the acetotrophic methanogens to the presence of LCFA. In another experiment, three recurrent pulses were performed with a real dairy wastewater containing 3.6 gCOD L(-1), from which 53% was fat. Methane yields of 0.45+/-0.01, 0.88+/-0.02 and 1.29+/-0.08 gCOD-CH(4) gCOD(fed)(-1) were achieved in the first, second and third pulses, respectively, evidencing an increasing capacity of the sludge to convert substrate accumulated in previous additions.     

Anaerobic degradation of sulphite evaporator condensate in a fixed-bed loop reactor by a defined bacterial consortium

Summary After elucidating the composition of an anaerobic bacterial enrichment culture treating sulphite evaporator condensate (SEC), an effluent in the pulp and paper industry, we built up stepwise a defined mixed culture to convert the organic constituents of SEC (acetate, methanol, furfural) to methane and CO₂. In batch cultures Desulfovibrio furfuralis and Methanobacterium bryantii degraded furfural in the absence of sulphate via inter-species H₂ transfer yielding 0.42 mol methane and 1.87 mol acetate/mol furfural degraded. When Methanosarcina barkeri was added to this diculture, acetate was also transformed to methane yielding 0.93 mol methane/mol acetate converted. This consortium (D. furfuralis, Methanobacterium bryantii and Methanosarcina barkeri) degraded furfural in continuous culture (fixed-bed loop reactor) to 92%, but the conversion of acetate was only 67%. The conversion of acetate could be further improved to 86% by adding 10 mm sulphate to the medium. This resulted in a space time yield of 10.9 g chemical oxygen demand (COD)/1 per day for the overall conversion. With a consortium consisting of M. barkeri, Methanobrevibacter arboriphilus, Methanosaeta concilii and D. furfuralis, a synthetic SEC could be degraded at a space time yield of 13.35 g COD/1 per day. This defined culture degraded all the constituents of SEC at an efficiency of almost 90% compared to an enrichment culture under identical conditions.Offprint requests to: U. Ney     

Changes in product formation and bacterial community by dilution rate on carbohydrate fermentation by methanogenic microflora in continuous flow stirred tank reactor

Changes in product formation during carbohydrate fermentation by anaerobic microflora in a continuous flow stirred tank reactor were investigated with respect to the dilution rate in the reactor. In the fermentation by methanogenic microflora, stable methane fermentation, producing methane and carbon dioxide, was observed at relatively low dilution rates (less than 0.33 d(-1) on glucose and 0.20 d(-1) on cellulose). Decomposition of cellulose in the medium was a rate-limiting step in the reaction, because glucose was easily consumed at all applied dilution rates (0.07-4.81 d(-1)). Intermediate metabolites of methane fermentation, such as lactate, ethanol, acetate, butyrate, formate, hydrogen, and carbon dioxide, were accumulated as dilution rate increased. Maximum yield of hydrogen was obtained at 4.81 d(-1) of dilution rate (0.1 mol/mol glucose on glucose or 0.7 mol/mol hexose on cellulose). Lactate was the major product on glucose (1.2 mol/mol glucose), whereas ethanol was predominant on cellulose (0.7 mol/mol hexose). An analysis by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial 16S rDNA of the microflora indicated that changes in the microbial community took place at various dilution rates, and these changes appeared to correspond to the changes in product distributions. Sequence analyses of the DGGE fragments revealed the probable major population of the microflora. A band closely related to the microorganisms of thermophilic anaerobic bacteria was detected with strong intensity on both glucose and cellulose. Differences in the production yield of hydrogen could have been caused by different populations of microorganisms in each microflora. In the case of cellulose, increasing the dilution rate brought about an accumulation of microorganisms related to Clostridia species that have cellulolytic activity, this being in accordance with the notion of cellulose decomposition being the rate-limiting reaction.     

Effect of ammonia on the methanogenic activity of methylaminotrophic methane producing Archaea enriched biofilm

Ammonia is a metabolic product in the decomposition of protein wastes, and has a recognized inhibitory effect on methanogenesis; this effect has been slightly quantified on methanogenic biofilms and particularly those populated by methanogenic Archaea which produce ammonia as a catabolic product from methylated amines. This paper presents studies on the effect of ammonia on maximum methanogenic activity of anaerobic biofilms enriched by methylaminotrophic methane producing Archaea (mMPA). The effect of unionized free ammonia on the specific maximum methanogenic activity of a mMPA enriched biofilm was studied, using 250 mL flasks containing ceramic rings colonized by 30 day-old experimental biofilm and adding 48.8 (control system), 73.8, 98.8, 148.8, 248.8, 448.8 and 848.8 mg NH(3)-N/L. The systems were maintained for ten days at a pH of 7.5 and temperature of 37 degrees C. The results showed that at 848.8 mg NH(3)-N/L, biofilm methane production required 36 h adaptation period, prior to entering into maximum production phase. The highest maximum methanogenic activity reached a value of 2.337+/-0.213 g COD methane/g VSS *day when 48.8 mg NH(3)-N/L was added, and inhibition was clearly observed in those systems above 148.8 mg NH(3)-N/L, producing under 1.658+/-0.185 g COD methane/g VSS *day. The lowest methanogenic activity reached was 0.639+/-0.162 g COD methane/g VSS *day at the system added with 848.8 mg NH(3)-N/L. When applying the Luong and non-competitive inhibition models, the best fit was obtained with the non-competitive model, which predicted 50% inhibition of methanogenic activity at 365.288 mg NH(3)-N/L.     

Methane recovery from water hyacinth through whole-cell immobilization technology

The concepts of feed pretreatment, phase separation, and whole-cell immobilization technology have been incorporated in this investigation for the development of rational and cost-effective two- and three-stage methane recovery systems from water hyacinth (WH)Analyses of laboratory data reveal that a three-stage system could be designed with an alkali pretreatment stage [3.6% Na(2)CO(3) + 2.5% Ca(OH)(2) W/W, 24 h HRT] followed by an open acid reactor (2.1 days HRT) and closed immobilized methane reactor (12 h HRT), providing steady-state COD conversion of 62-65%, TVA conversion of 91-95%, and gas productivity of 4.08-5.36 L/L reactor volume/day with 82% methane. A gas yield of 50 L/kg WH/day (dry wt basis) at 35-37 degrees C is possible with this system. Insulation bricks, with particle size distribution of 500-3000 mum, were used as support material in the reactors at organic loading rate of 20 kg COD/m(3) day. The reactors matured in 15-18 weeksSubstantial reduction in retention time for the conversion of volatile acids in immobilized methane reactors prompted further research on the combined immobilized reactor to make possible an additional reduction in the cost of a WH-based biogas system. Evaluation of laboratory data reveals that a two-stage system could be designed with an open alkali pretreatment stage and a combined immobilized reactor (12 h HRT), providing steady-state COD conversion of 53% and gas productivity of 3.1 L/L reactor volume/day with 86% methane. A gas yield of 44 L/kg WH/day (dry wt basis) at 35-37 degrees C could be obtained from this system. Insulation bricks, with 500-1000 mum particle size distribution, was used as support material at an organic loading rate of 15 kg COD/m(3) day. Notwithstanding the fact that the technology in this study has been developed with water hyacinth as substrate, the implicit principles could be extended to any other organic substrate.     

Biodegradable polymers from organic acids by using activated sludge enriched by aerobic periodic feeding

This article describes a new process for the production of biopolymers (polyhydroxyalkanoates, PHAs) based on the aerobic enrichment of activated sludge to obtain mixed cultures able to store PHAs at high rates and yields. Enrichment was obtained through the selective pressure established by feeding the carbon source in a periodic mode (feast and famine regime) in a sequencing batch reactor. A concentrated mixture of acetic, lactic, and propionic acids (overall concentration of 8.5 gCOD L(-1)) was fed every 2 h at 1 day(-1) overall dilution rate. Even at such high organic load (8.5 gCOD L(-1) day(-1)), the selective pressure due to periodic feeding was effective in obtaining a biomass with a storage ability much higher than activated sludges. The immediate biomass response to substrate excess (as determined thorough short-term batch tests) was characterized by a storage rate and yield of 649 mgPHA (as COD) g biomass (as COD)(-1) h(-1) and 0.45 mgPHA (as COD) mg removed substrates (as COD(-1)), respectively. When the substrate excess was present for more than 2 h (long-term batch tests), the storage rate and yield decreased, whereas growth rate and yield significantly increased due to biomass adaptation. A maximum polymer fraction in the biomass was therefore obtained at about 50% (on COD basis). As for the PHA composition, the copolymer poly(beta-hydroxybutyrate/beta-hydroxyvalerate) with 31% of hydroxyvalerate monomer was produced from the substrate mixture. Comparison of the tests with individual and mixed substrates seemed to indicate that, on removing the substrate mixture for copolymer production, propionic acid was fully utilized to produce propionylCoA, whereas the acetylCoA was fully provided by acetic and lactic acid.     

Direct fermentation of fodder maize, chicory fructans and perennial ryegrass to hydrogen using mixed microflora

This study examined the feasibility of producing hydrogen by direct fermentation of fodder maize, chicory fructooligosaccharides and perennial ryegrass (Lolium perenne) in batch culture (pH 5.2-5.3, 35 degrees C, heat-treated anaerobically digested sludge inoculum). Gas was produced from each substrate and contained up to 50-80% hydrogen during the peak periods of gas production with the remainder carbon dioxide. Hydrogen yields obtained were 62.4+/-6.1mL/g dry matter added for fodder maize, 218+/-28mL/g chicory fructooligosaccharides added, 75.6+/-8.8mL H(2)/g dry matter added for wilted perennial ryegrass and 21.8+/-8mL H(2)/g dry matter added for fresh perennial ryegrass. Butyrate, acetate and ethanol were the main soluble fermentation products. Hydrogen yields of 392-501m(3)/hectare of perennial ryegrass per year and 1060-1309m(3)/hectare of fodder maize per year can be obtained based on the UK annual yield per hectare of these crops. These results significantly extend the range of substrates that can be used for hydrogen production without pre-treatment.     

Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor

A novel bioreactor containing self-flocculated anaerobic granular sludge was developed for high-performance hydrogen production from sucrose-based synthetic wastewater. The reactor achieved an optimal volumetric hydrogen production rate of approximately 7.3 L/h/L (7,150 mmol/d/L) and a maximal hydrogen yield of 3.03 mol H2/mol sucrose when it was operated at a hydraulic retention time (HRT) of 0.5 h with an influent sucrose concentration of 20 g COD/L. The gas-phase hydrogen content and substrate conversion also exceeded 40 and 90%, respectively, under optimal conditions. Packing of a small quantity of carrier matrices on the bottom of the upflow reactor significantly stimulated sludge granulation that can be accomplished within 100 h. Among the four carriers examined, spherical activated carbon was the most effective inducer for granular sludge formation. The carrier-induced granular sludge bed (CIGSB) bioreactor was started up with a low HRT of 4-8 h (corresponding to an organic loading rate of 2.5-5 g COD/h/L) and enabled stable operations at an extremely low HRT (up to 0.5 h) without washout of biomass. The granular sludge was rapidly formed in CIGSB supported with activated carbon and reached a maximal concentration of 26 g/L at HRT = 0.5 h. The ability to maintain high biomass concentration at low HRT (i.e., high organic loading rate) highlights the key factor for the remarkable hydrogen production efficiency of the CIGSB processes.     

Treatment of molasses wastewater by acetogenic bacteria BP103 in sequencing batch reactor (SBR) system

Acetogenic bacteria BP103 cells could be used as the absorbent for melanoidin pigment (MP) and molasses wastewater (MWW). The maximum MP adsorption yield of this strain observed from the dead (autoclaved) cell. It was two times higher than that with resting cells. However, the MP adsorption yield of the strain was 50-60% decreased by acclimatization with the media containing MP. The deteriorated cells (MP-adsorbed cells) could be recovered by washing with 0.1% SDS, 0.1% Tween 80 and 0.1 mol/L NaOH solutions. Among them, 0.1 mol/L NaOH solution was most suitable according to highest elution ability and no-effect to the MP adsorption capacity (The adsorption yield of deteriorated cell was reduced only 10% after washing three times with 0.1 mol/L NaOH solution). In SBR system, the strain showed very low MP removal yield with both molasses wastewater (MWW) from the anaerobic pond (An-MWW) and stillage from an alcohol factory (U-MWW). However, the MP removal yield was increased by supplementation with carbon sources (glucose). Also, the MP removal efficiency was increased with the increase of supplemented-glucose concentration. The highest COD, BOD(5), TKN and MP removal efficiencies of the SBR system with 10 times-diluted An-MWW solution containing 30 g/L glucose under HRT of seven days were 65.2+/-2.5%, 82.8+/-3.4%, 32.1+/-0.8% and 50.2+/-3.7%, respectively. The large molecular weight fraction of MP in both U-MWW and An-MWW solutions were rapidly removed by acetogenic bacteria BP103, while the small molecular weight fractions of MP still remained in the effluent.     

Carbon and nitrogen substrates consumption, ammonia release and proton transfer in relation with growth of Geotrichum candidum and Penicillium camemberti on a solid medium

The influence of hydraulic retention time (HRT) and gelatin concentration on the acidification of gelatinaceous wastewater in an upflow anaerobic reactor was investigated at pH 5.5 and 37 degrees C. The degree of gelatin degradation increased with the HRT, from 84.1% at 4 h to 89.6% at 24 h, but decreased with the increase of the gelatin concentration in the influent from 65.2% at 2 g-CODl(-1) to 51.9% at 30 g-CODl(-1). The degradation of gelatin followed the Monod kinetics with a maximum rate of 1.10 g (g-VSS x d)(-1) and a half-rate constant of 0.23 gl(-1). The overall production rate of VFA and alcohols decreased with HRT, from 0.33 g (g-VSS x d)(-1) at 4 h to 0.15 g (g-VSS x d)(-1) at 24 h, but increased with gelatin concentration in the influent, from 0.10 g (g-VSS x d)(-1) at 4 g-CODl(-1) to 0.58 g (g-VSS x d)(-1) at 30 g-CODl(-1). The key acidification products were acetate, propionate and butyrate, plus i-butyrate, valerate, i-valerate, caproate and ethanol in smaller quantities. Formate, methanol, propanol and butanol were found only in certain runs. Only 4.5-7.8% of COD in wastewater was converted to hydrogen and methane. The sludge yield was estimated as 0.320+/-0.014 g-VSS (g-COD)(-1).     

Effect of various factors on ethanol yields from lignocellulosic biomass by Thermoanaerobacterium AK₁₇

The ethanol production capacity from sugars and lignocellulosic biomass hydrolysates (HL) by Thermoanaerobacterium strain AK(17) was studied in batch cultures. The strain converts various carbohydrates to, acetate, ethanol, hydrogen, and carbon dioxide. Ethanol yields on glucose and xylose were 1.5 and 1.1 mol/mol sugars, respectively. Increased initial glucose concentration inhibited glucose degradation and end product formation leveled off at 30 mM concentrations. Ethanol production from 5 g L(-1) of complex biomass HL (grass, hemp, wheat straw, newspaper, and cellulose) (Whatman paper) pretreated with acid (0.50% H(2) SO(4)), base (0.50% NaOH), and without acid/base (control) and the enzymes Celluclast and Novozyme 188 (0.1 mL g(-1) dw; 70 and 25 U g(-1) of Celluclast and Novozyme 188, respectively) was investigated. Highest ethanol yields (43.0 mM) were obtained on cellulose but lowest on hemp leafs (3.6 mM). Chemical pretreatment increased ethanol yields substantially from lignocellulosic biomass but not from cellulose. The influence of various factors (HL, enzyme, and acid/alkaline concentrations) on end-product formation from 5 g L(-1) of grass and cellulose was further studied to optimize ethanol production. Highest ethanol yields (5.5 and 8.6 mM ethanol g(-1) grass and cellulose, respectively) were obtained at very low HL concentrations (2.5 g L(-1)); with 0.25% acid/alkali (v/v) and 0.1 mL g(-1) enzyme concentrations. Inhibitory effects of furfural and hydroxymethylfurfural during glucose fermentation, revealed a total inhibition in end product formation from glucose at 4 and 6 g L(-1), respectively.     

Effect of high nitrate concentration on PHB storage in sequencing batch reactor under anoxic conditions

The study investigated effect of high influent nitrate concentration on poly-beta-hydroxybutyrate, (PHB), storage in a sequencing batch reactor, (SBR), under anoxic conditions. Acetate was fed as pulse during anoxic phase, sustained with external nitrate feeding. SBR operation involved three runs at steady state with COD/N ratios of 3.84, 2.93 and 1.54 gCOD/gN, where external nitrate concentrations gradually increased from 50 mg N/l to 114 mg N/l and 226 mg N/l, in 1st, 2nd and 3rd runs, respectively. In 1st run, acetate was fully converted into PHB with the storage yield value of 0.57-0.59 gCOD/gCOD, calculated both in terms of PHB formation and NO(X) utilization, confirming storage was the sole substrate utilization mechanism. In the following runs, PHB formation was reduced and the storage yield based on PHB dropped down to 0.40 and 0.33 gCOD/gCOD with increasing influent nitrate concentrations, indicating that higher portions of acetate were diverted to simultaneous direct growth. The observations suggested that nitrite accumulation detected at low COD/N ratios was responsible for inhibition of PHB storage.     

Acclimation Strategy of a Biohydrogen Producing Population in a Continuous‐Flow Reactor with Carbohydrate Fermentation

Poor startup of biological hydrogen production systems can cause an ineffective hydrogen production rate and poor biomass growth at a high hydraulic retention time (HRT), or cause a prolonged period of acclimation. In this paper a new startup strategy was developed in order to improve the enrichment of the hydrogen‐producing population and the efficiency of hydrogen production. A continuously‐stirred tank reactor (CSTR) and molasses were used to evaluate the hydrogen productivity of the sewage sludge microflora at a temperature of 35 °C. The experimental results indicated that the feed to microorganism ratio (F/M ratio) was a key parameter for the enrichment of hydrogen producing sludge in a continuous‐flow reactor. When the initial biomass was inoculated with 6.24 g of volatile suspended solids (VSS)/L, an HRT of 6 h, an initial organic loading rate (OLR) of 7.0 kg chemical oxygen demand (COD)/(m³ × d) and an feed to microorganism ratio (F/M) ratio of about 2–3 g COD/(g of volatile suspended solids (VSS) per day) were maintained during startup. Under these conditions, a hydrogen producing population at an equilibrium state could be established within 30 days. The main liquid fermentation products were acetate and ethanol. Biogas was composed of H₂ and CO₂. The hydrogen content in the biogas amounted to 47.5 %. The average hydrogen yield was 2.01 mol/mol hexose consumed. It was also observed that a special hydrogen producing population was formed when this startup strategy was used. It is supposed that the population may have had some special metabolic pathways to produce hydrogen along with ethanol as the main fermentation products.     

Methane recovery from water hyacinth through anaerobic activated sludge process

The concepts of phase separation, anaerobic activated sludge process, and alkali pretreatment have been incorporated in this investigation with the objective of developing rational and cost-effective designs of diphasic anaerobic activated sludge systems, with and without alkali treatment, for methane recovery from water hyacinth (WH). Evaluation of process kinetics and optimization analyses of laboratory data reveal that a diphasic system with alkali treatment could be designed with an alkali pretreatment step (3.6% Na(2)CO(3) + 2.5% Ca(OH)(2) (w/w) of WH, 24 h duration) followed by an open acid phase (2.1 days HRT) and closed methane reactor with sludge recycle (5.7 days HRT, 7.7 days MCRT) for gas yield of 50 L/kg WH/d at 35-37 degrees C. Likewise, a diphasic system without alkali treatment could be designed with an open acid phase (2 days HRT) followed by closed methane reactor with sludge recycle (3.2 days HRT, 6 days MCRT) for gas yield of 32.5 L/kg WH/d at 35-37 degrees C. Detailed economic analyses bring forth greater cost-efficacy of the diphasic system without alkali treatment and reveal that the advantage accrued in terms of higher gas yield is overshadowed by the cost of chemicals in the diphasic system with alkali treatment.     

Increased digestibility of bagasses by pretreatment with alkalis and steam explosion

Alkali treatment and steam explosion of bagasse were investigated in order to develop economical and effective methods of increasing the digestibility of bagasse. The treated bagasse was to be used as a substrate for the production of volatile fatty acids by anaerobic acidogenic bacteria. The alkalis examined were NaOH, NH(3) (aqueous), NaOH + NH(3), Ca(OH)(2), and Ca(OH)(2) + Na(2)CO(3), at ambient temperature and in combination with steam explosion at 200 degrees C, 6.9 MPa, and 5 min cooking times. Digestibilities of up to 733 g organic matter (OM)/kg bagasse dry matter (DM) were obtained for bagasse treated with NaOH and Ca(OH)(2) + Na(2)CO(3); less than 430 g OM was obtained for bagasse treated with aqueous NH(3); and up to 724 g OM was obtained for bagasse treated with Ca(OH)(2). This digestibility was only achieved by using high concentrations of Ca(OH)(2), i.e., 180-300 g/kg bagasse. Steam explosion increased the digestibility of bagasse up to 740 g OM in the presence of alkali but only to 610 g OM in the absence of alkali. The digestibility of bagasse without pretreatment was 190 g OM/kg bagasse DM. More than one-half the hemicellulose present was solubilized by pretreatment. The composition of the liquid fraction of steam-exploded material was examined and contained mainly xylose monomers and oligomers (112 g/kg original bagasse DM) and acetic acid (33 g/kg original DM). The relative costs of the alkalis used were obtained for the United States, Australia, and Europe. Lime [Ca(OH)(2)] was the least expensive alkali per unit of additional digestible OM obtained. Ammonia was the most expensive alkali to use, except in the United States where the difference in its cost relative to other alkalis was smaller. However, ammonia provides organic nitrogen for microbial growth, and could be recycled. With acidogenic fermentations, alkali is able to double as a neutralizing agent during fermentation. Thus, concentrations of alkali equal to that required for neutralization may be used in pretreatment. Concentrations of Ca(OH)(2) as high as 300 g/kg bagasse were needed for neutralization and should, therefore, be considered for pretreatment. Steam explosion of bagasse resulted in digestible, sterilized substrates of small particle size with readily separable liquid and pulp streams.     

Fermentative hydrogen production and bacterial community structure in high-rate anaerobic bioreactors containing silicone-immobilized and self-flocculated sludge

A novel continuously stirred anaerobic bioreactor (CSABR) seeded with silicone-immobilized sludge was developed for high-rate fermentative H2 production using sucrose as the limiting substrate. The CSABR system was operated at a hydraulic retention time (HRT) of 0.5-6 h and an influent sucrose concentration of 10-40 g COD/L. With a high feeding sucrose concentration (i.e., 30-40 g COD/L) and a short HRT (0.5 h), the CSABR reactor produced H2 more efficiently with the highest volumetric rate (VH2) of 15 L/h/L (i.e., 14.7 mol/d/L) and an optimal yield of ca. 3.5 mol H2/mol sucrose. The maximum VH2 value obtained from this work is much higher than any other VH2 values ever documented. Formation of self-flocculated granular sludge occurred during operation at a short HRT. The granule formation is thought to play a pivotal role in the dramatic enhancement of H2 production rate, because it led to more efficient biomass retention. A high biomass concentration of up to 35.4 g VSS/L was achieved even though the reactor was operated at an extremely low HRT (i.e., 0.5 h). In addition to gaining high biomass concentrations, formation of granular sludge also triggered a transition in bacterial community structure, resulting in a nearly twofold increase in the specific H2 production rate. According to denatured-gradient-gel-electrophoresis analysis, operations at a progressively decreasing HRT resulted in a decrease in bacterial population diversity. The culture with the best H2 production performance (at HRT = 0.5 h and sucrose concentration = 30 g COD/L) was eventually dominated by a presumably excellent H2-producing bacterial species identified as Clostridium pasteurianum.     

Continuous fermentative hydrogen production from a wheat starch co-product by mixed microflora

For the transition to the hydrogen economy, hydrogen must be produced sustainably, e.g., by the fermentation of agricultural material. Continuous fermentative production of hydrogen from an insoluble substrate in nonsterile conditions is yet to be reported. In this study hydrogen production using mixed microflora from heat-treated digested sewage sludge in nonsterile conditions from a particulate co-product of the wheat flour industry (7.5 g L(-1) total hexose) at 18- and 12-hour hydraulic retention times, pH 4.5 and 5.2, 30 degrees C and 35 degrees C was examined. In continuous operation, hydrogen yields of approximately 1.3 moles hydrogen/mole hexose consumed were obtained, but decreased if acetate or propionate levels rose, indicating metabolism shifted towards hydrogen consumption by homoacetogenesis or propionate producers. These shifts occurred both at pH 4.5 and 5.2. Sparging the reactor with nitrogen to reduce hydrogen in the off-gas from 50% to 7% gave stable operation with a hydrogen yield of 1.9 moles hydrogen /mole hexose consumed over an 18-day period.     

Influence of dilution and phase separation on the anaerobic digestion of olive mill wastewaters

The high organic content of Olive Mill Wastewaters (OMW) causes some difficulties in maintaining the anaerobic process efficiency at high level. The two phase anaerobic system was used to treat olive mill wastewaters, diluted with tap water. Phase separation was accomplished through control of the hydraulic retention time and initial COD removal in two reactors operated in series. The effect of substrate concentration and phase separation on removal efficiency has been investigated. Experimental results indicated that yield of 0.322 to 0.335 litre biogas/g COD removal were obtained with two phase anaerobic treatment and space loading rate of 2.3 and 2.4 gCOD/l.day. The maximum methane production rate near to the theoretical value and corresponded to 360 ml of CH₄ for 1g COD removal.Abbreviations OMW Olive Mill Wastewaters - VFA Volatile Fatty Acid - COD Chemical Oxygen Demand - HRT High Retention Time - TKN Total Kjeldahl Nitrogen