A combined pig model to determine the net absorption of volatile fatty acids in the large intestine under different levels of crude fiber

Background:This study aimed to develop a combined model to quantify the net absorption of volatile fatty acids(VFA)in the large intestine(LI)of pigs.Methods:Fifteen female growing pigs(Duroc×Large White×Landrace)were ranked by body weight(30±2.1 kg)on day 0 and assigned to one of three treatments,namely the basal diet containing different crude fiber(CF)levels(LCF:3.0%CF,MCF:4.5%CF,and HCF:6.0%CF).The pigs were implanted with the terminal ileum fistula and the cannulation of the ileal mesenteric vein(IMV),portal vein(PV),and left femoral artery(LFA)from days 6 to 7.[13 C]-Labeled VFA and P-aminohippuric acid were constantly perfused into the terminal ileum fistula and the cannulation of the IMV(day 15),respectively.Blood samples were collected from the PV and the LFA during perfusion(5 h),and LI samples were collected.Results:The net flux of[12 C]-acetic acid in the PV was greater for LCF versus MCF(p=0.045),but no difference was observed in the net flux of[12 C]-propionic acid(p=0.505)and[12 C]-butyric acid(p=0.35)in the PV among treatments.The deposition of[12 C]-acetic acid in the LI was greater for LCF versus MCF(p=0.014),whereas the deposition of[12 C]-propionic acid(p=0.007)and[12 C]-butyric acid(p=0.037)in the LI was greater for LCF versus HCF.Conclusions:In conclusion,this pig model was found conducive to study the net absorption of VFAs in the LI,and LCF had more net absorption of VFAs in the LI than MCF and HCF.

Microbial Community Profiles Related to Volatile Fatty Acids Production in Mesophilic and Thermophilic Fermentation of Waste Activated Sludge Pretreated by Enzymolysis

Mesophilic and thermophilic anaerobic fermentation performance of waste activated sludge(WAS)pretreated by enzymes catalysis associated with microbial community shifts were investigated.WAS disintegration was boosted considerably by enzymolysis with 8750 mg/L of soluble COD release within 180 min.Mesophilic anaerobic fermentation(MAF)produced nearly equal VFA accumulation with over 3200 mg COD/L compared with that of thermophilic fermentation(TAF).Bacterial community consortia showed great shifting differences in dynamics of main T⁃RFs between MAF and TAF.Moreover,MAF was conducive to form intermediate bacterial community evenness compared to TAF,which preserved a robust function of VFA production.The enzymes catalysis prompted bio⁃energy(electricity)recovery potential of WAS organics via anaerobic fermentation(MAF/TAF)with evaluating electricity conversion efficiency of 0.75-0.82 kW·h/kg VSS(3.9 times higher than control test).Finally,this study proposed some novel thinking on future WAS treatment/management towards energy recovery coupled with energy⁃sufficient wastewater treatment by co⁃locating WAS anaerobic fermentation,MFC plant with wastewater treatment plant(s).

Linkages of volatile fatty acids and polyhexamethylene guanidine stress during sludge fermentation:Metagenomic insights of microbial metabolic traits and adaptation

The massive use of polyhexamethylene guanidine(PHMG),as a typical bactericidal agent,raised environmental concerns to the public.This work comprehensively revealed the hormesis effects of PHMG occurred in waste activated sludge(WAS)on the generation of volatile fatty acids(VFAs)during anaerobic fermentation.The low level of PHMG(100 mg/g TSS)significantly promoted the VFAs generation(1283 mg COD/L,compared with 337 mg COD/L in the control)via synchronously facilitating the solubilization,hydrolysis,and acidification steps but inhibiting methanogenesis.Metagenomic analysis showed that the functional anaerobe(i.e.,Bacteroides,Macellibacteroide and Parabacteroide)and corresponding genetic expressions responsible for extracellular hydrolysis(i.e.,clp P),membrane transport(i.e.,ffh and gsp F),intracellular substrates metabolism(i.e.,ald and paa F)and VFAs biosynthesis(i.e.,ACACA and FASN)were enhanced in the optimal presence of PHMG.Moreover,the anaerobic species could respond and adapt to low PHMG stimuli via quorum sensing(i.e.,cqs A,rpf C and rpf G),and thus maintain the high microbial metabolic activities.However,they were unable to tolerate the toxicity of excessive PHMG,resulting in the extremely low VFAs production.This work enlightened the effects of emerging pollutants on WAS fermentation at the genetic levels,and provided guidance on the WAS treatment and resource recovery.

Volatile fatty acids carbon source for biological denitrification

In the suspended sludge system, when pH and temperature were suitable and carbon source was not limited, the denitrification and C-uptake rate followed a zero-order reaction. Mixed VFA C-source had higher denitrification rate than single VFA C-source consisting of them. When VFA were used as carbon source, consumed carbon quantity for denitrification was closed to 1. 07 mg VFA-C/mg NO-N. About 20% of the applied C/N was used for assimilative purposes. As rising up influent C/N by increasing the C load ,the sludge yield YN increased. The part of carbon consumption increased and the effluent oxidized nitrogen decreased. At pH 7. 5, 25℃ and sludge yield 0.72 mg VSS/mg NO-N, the calculated influent VFA-C /NO critical value was 1. 43 for complete denitrification.

Effects of sludge age on anaerobic acidification of waste activated sludge: Volatile fatty acids production and phosphorus release

Effects of sludge age on volatile fatty acids (VFAs) production and Phosphorus (P) release during anaerobic acidification of waste activated sludge (WAS) were investigated.Sequencing batch reactors (SBR) fed with simulating domestic sewage were applied to produce WAS of different sludge ages,and batch tests were used for anaerobic acidification.The maximum dissolved total organic carbon,release of PO_(4)^(3+)-P,and accumulation of acetate (C2),propionate (C3),butyrate (C4),and valerate (C5) decreased by 56.2%,55.8%,52.6%,43.7%,82.4%and 84.8%,respectively,as the sludge age of WAS increased from 5 to 40 days.Limited degradation of protein played a dominating role in decreasing DTOC and VFAs production.Moreover,the increase in molecular weight of organics and organic nitrogen content in the supernatant after acidification suggested that the refractory protein in WAS increased as sludge age extended.Although the production of C2,C3,C4,and C5 from WAS decreased as the sludge age increased,the proportions of C2 and C3 in VFAs increased,which might be due to the declined production of C5 from protein and the faded genus Dechlorobacter.Keeping sludge age of WAS at a relatively low level (<10 days) is more appropriate for anaerobic acidification of WAS as internal carbon sources and P resource.

Enhanced nitrogen removal upon the addition of volatile fatty acids from activated sludge by combining calcium peroxide and low-thermal pretreatments

This study investigated a combined low-thermal and CaO_(2)pretreatment to enhance the volatile fatty acid(VFA)production from waste activated sludge(WAS).The fermentative product was added to a sequencing batch reactor(SBR)as an external carbon source to enhance nitrogen removal.The results showed that the combined pretreatment improved WAS solubilization,releasing more biodegradable substrates,such as proteins and polysaccharides,from TB-EPS to LB-EPS and S-EPS.The maximum VFA production of 3529±188 mg COD/L was obtained in the combined pretreatment(0.2 g CaO_(2)/g VS+70℃for 60 min),which was 2.1 and 1.4-fold of that obtained from the sole low-thermal pretreatment and the control test,respectively.Consequently,when the fermentative liquid was added as an external denitrification carbon source,the effluent total nitrogen decreased to Class A of the discharge standard for pollutants in rural wastewater treatment plants in most areas of China.

A novel online analyzer for accurate and rapid measurement of volatile fatty acids in anaerobic wastewater treatment

Volatile fatty acids(VFAs), which are largely generated during the anaerobic acidification process, are considered to be reliable indicators of the stable process operation. However,the common methods for monitoring VFAs are offline, and they are typically manual requiring time-consuming, costly and complex instruments. This study aims to develop a novel online analyzer for automatic measuring VFAs, which was based on the 5-p H point titration, embedded with a proportional-integral-derivative(PID) feedback control system. The results show that it can achieve accurate and rapid monitoring of VFAs ranging between 0-400 mg/L(<9 min/sample) but simultaneously faces the problems of overtitration and interference of complex characteristics of wastewater. In order to improve its accuracy and stability, the effects of three general coefficients(K_(I),K_(P), and K_(D)) of PID on the titration were investigated, and the optimal values of K_(I), K_(P), and K_(D)were found to be 1.5, 1.0, and-1.0~0.5,respectively. Besides, the initial titration speed was set at 0.06 m L/min, equal to the minimum speed of the peristaltic pump, and the dichotomy approach was integrated into the PID feedback controller. Owing to the above improvements, the relative mean deviation and standard deviation of measuring VFAs in both synthetic and real wastewaters were mostly lower than 5.0% and 5.0 mg/L, proving the online analyzer is rapid, accurate and reliable.

Dietary pectic oligosaccharide supplementation improves rat reproductive performance via regulating intestinal volatile fatty acids during middle gestation

As a kind of green additive,pectic oligosaccharide(POS) may regulate some physiological functions of animals,such as gut health,antioxidant capacity,immunity and lipid metabolism.This study aimed to identify whether POS administration can improve maternal reproduction,and to determine the possible metabolism.A total of 48 pregnant Wistar rats randomly allotted into 2 groups,and each group was fed a diet supplemented with 0 or 800 mg/kg of POS.Pectic oligosaccharide administration increased rat born number(P <0.05),did not affect rat embryo number on d 7 of gestation,but increased rat fetus number on d 14 of gestation(P <0.05).On d 14 of gestation,POS treatment improved Lactobacillus and Bifidobacterium populations and volatile fatty acid concentrations of cecal digesta(P <0.05),hormone(progesterone and nitric oxide) and cytokine(interleukin 2) concentrations of serum(P <0.05),and antioxidant capacity of serum(increased total antioxidant capacity and decreased malondialdehyde) and placenta(increased total superoxide dismutase,decreased malondialdehyde)(P <0.05) in pregnant rats.These results suggest that POS administration improved rat reproduction via decreasing fetus loss in middle gestation.This was due to the increased volatile fatty acid concentrations in rat gut improving hormone and inflammatory-cytokine productions,and antioxidant capacity.

Impact of undissociated volatile fatty acids on acidogenesis in a two-phase anaerobic system

This study investigated the degradation and production of volatile fatty acids（VFAs）in the acidogenic phase reactor of a two-phase anaerobic system.20 mmol/L bromoethanesulfonic acid（BESA）was used to inhibit acidogenic methanogens（which were present in the acidogenic phase reactor）from degrading VFAs.The impact of undissociated volatile fatty acids（un VFAs）on＂net＂VFAs production in the acidogenic phase reactor was then evaluated,with the exclusion of concurrent VFAs degradation.＂Net＂VFAs production from glucose degradation was partially inhibited at high un VFAs concentrations,with 59%,37% and 60% reduction in production rates at 2190 mg chemical oxygen demand（COD）/L undissociated acetic acid（un HAc）,2130 mg COD/L undissociated propionic acid（un HPr）and 2280 mg COD/L undissociated n-butyric acid（un HBu）,respectively.The profile of VFAs produced further indicated that while an un VFA can primarily affect its own formation,there were also un VFAs that affected the formation of other VFAs.

Impacts of the unsaturation degree of long-chain fatty acids on the volatile fatty acid profiles of rumen microbial fermentation in goats in vitro

This study investigated the impacts of the degree of unsaturation （unsaturity） of long-chain fatty acids on volatile fatty acid （VFA） profiles of rumen fermentation in vitro. Six types of long-chain fatty acids, including stearic acid （C18：0, control group）, oleic acid （C18：1, n-9）, linoleic acid （C18：2, n-6）, a-linolenic acid （C18：3, n-3）, arachidonic acid （C20：4, n-6） and eicosapentaenoic acid （C20：5, n-3）, were tested. Rumen fluid from three goats fitted with ruminal fistulae was used as inoculum and the inclusion rate of long-chain fatty acid was at 3% （w/w） of substrate. Samples were taken for VFA analysis at 0, 3, 6, 9, 12, 18 and 24 h of incubation, respectively. The analysis showed that there were significant differences in the total VFA among treatments, sampling time points, and treatment×time point interactions （P〈0.01）. a-Linolenic acid had the highest total VFA （P〈0.01） among different long-chain fatty acids tested. The molar proportion of acetate in total VFA significantly differed among treatments （P〈0.01） and sampling time points （P〈0.01）, but not treatment×time point interactions （P〉0.05）. In contrast, the molar proportion of propionate did not differ among treatments during the whole incubation （P〉0.05）. However, for butyrate molar proportions, significant differences were found not only among sampling time points but also among treatments and treatment×time point interactions （P〈0.01）, with eicosapentaenoic acid having the highest value （P〈0.01）. Additionally, no statistically significant differences were found in the acetate to propionate ratios among treatments groups （P〉0.05）, even the treatments stearic acid and a-linolenic acid were numerically higher than the others. The inclusion of 3% long-chain unsaturated fatty acids differing in the degree of unsaturation brought out a significant quadratic regression relation between the total VFA concentration and the double bond number of fatty acid. In conclusion, the a-linolenic acid with 3 double bonds appeared better for improving rumen microbial fermentation and the total VFA concentration.

Effects of Ultrasonic and Acid Pretreatment on Food Waste Disintegration and Volatile Fatty Acid Production

This study aims at investigating the effects of ultrasonic and acid pretreatment on food waste( FW)disintegration and volatile fatty acid( VFA) production. Single-factor experiments are carried out to obtain optimal conditions of individual ultrasonic and acid pretreatment,and response surface method( RSM) is applied to optimize the conditions of the combination of ultrasonic and acid( UA) pretreatment. Results show that the optimal acid,ultrasonic and UA pretreatments conditions are individual pH 2,individual ultrasonic energy density of 1. 0 W / mL and the combination of ultrasonic energy density1. 11 W / mL and pH 1. 43,respectively. Correspondingly,the maximum disintegration degrees( DD) of 46. 90%,57. 38% and68. 83%are obtained by acid,ultrasonic and UA pretreatments,respectively. After optimizing pretreatment conditions,batch experiments are operated to produce VFA from raw and pretreated FW under anaerobic fermentation process. Both the maximum VFA production( 976. 17 mg COD / gV S) and VFA / SCOD( 72. 89%) are obtained with ultrasonic pretreatment, followed by UA pretreatment, non-pretreatment and acid pretreatment,respectively. This observation demonstrates that a higher acidity on acid and UA pretreatments inhibits the generation of VFA. Results suggest that ultrasonic pretreatment is preferable to promote the disintegration degree of FW and VFA production.

Processing Effect on the Physicochemical and Volatile Fatty Acid Profile of African Breadfruit (Treculia africana) Seed Oil

African breadfruit seeds were subjected to three processing methods—parboiling, cooking and toasting, and the raw was used as control. The purpose of this research was to extract the oil from the seed and to determine the effect of processing on the oil for physicochemical properties and volatile fatty acid profile. Physicochemical properties showed that the colour of the oil varied from golden yellow to brownish yellow with specific gravity varying between 0.802 g/cm3 and 0.813 g/cm3. Percentage yield of oil was 6.14% for raw extract, 6.62% for parboiled extract, 7.56% for toasted extract, and 5.01% for cooked extract. Acid, peroxide and saponification value for oil extracted from the raw seed varied with the processed samples value. The Volatile Fatty Acid (VFA), also known as Short Chain Fatty Acid (SCFA) found inherent in varying concentration, were formic, acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocarproic, hexanoic and heptanoic acid. Overall results prove that heat results in increases in the VFA concentration of the processed oil.

Evaluation of in vitro digestibility of Aspergillus oryzae fungal biomass grown on organic residue derived‑VFAs as a promising ruminant feed supplement

Background As demand for high quality animal feed continues to raise,it becomes increasingly important to mini-mize the environmental impact of feed production.An appealing sustainable approach to provide feed fractions is to use organic residues from agro-food industry.In this regard,volatile fatty acids(VFAs)such as acetic,propionic and butyric acids,derived from bioconversion of organic residues can be used as precursors for production of micro-bial protein with ruminant feed inclusion potential.This study aims to investigate the in vitro digestibility of the Asper-gillus oryzae edible fungal biomass cultivated on VFAs-derived from anaerobic digestion of residues.The produced fungal protein biomass,along with hay clover silage and rapeseed meal were subjected to various in vitro assays using two-stage Tilley and Terry(TT),gas,and bag methods to evaluate and compare its digestibility for application in ruminant feed.Results The produced fungal biomass contained a higher crude protein(CP)(41%–49%)and rather similar neutral detergent fiber(NDF)(41%–56%)compared to rapeseed meal.The rumen in vitro dry matter digestibility(IVDMD)of the fungal biomass in the TT method ranged from 82%to 88%(statistically similar to that of the gas method(72%to 85%)).The IVDMD of fungal biomass were up to 26%and 40%greater than that of hay clover silage and rapeseed meal,respectively.The type of substrate and bag method had pronounced effect on the fermentation products(ammonium-N(NH4+-N),total gas and VFAs).Fungal biomass digestion resulted in the highest release of NH4+-N(340–540 mg/L)and the ratio of acetate to propionate ratio(3.5)among subjected substrates.Conclusion The results indicate that gas method can be used as a reliable predictor for IVDMD as well as fermenta-tion products.Furthermore,the high IVDMD and fermentation product observed for Aspergillus oryzae fungal biomass digestion,suggest that the supplementation of fungal biomass will contribute to improving the rumen digestion by providing necessary nitrogen and energy to the ruminant and microbiota.

CHARACTERISTICS OF THERMOPHILIC ANAEROBIC ACIDOGENESIS OF STARCH WASTEWATER

Acidogenic dissimilation of synthetic starch wastewater (1 000～10 000 mg COD·L -1 ) was studied in a thermophilic (55 ℃) upflow anaerobic sludge blanket (UASB) reactor.The production of volatile fatty acids (VFA) was proportional to the chemical oxygen demand (COD) loading rate.The yield of VFA was around 0.28 g VFA/g COD over the COD loading rate from 1.25 to 30 g COD·L -1 ·d -1 and the hydraulic retention time from 8.8 h to 24 h.Distribution of organic acids,the contents of propionic and butyric acids in the effluent in particular were also dependent on the COD loading rate.The thermophilic UASB reactor showed a stable performance on hydrolysis and acidogenesis of starch as well as suspended solid removal at short hydraulic retention times and high influent pH(10～11),during the operation of 110 d.

Releasing characteristics of phosphorus and other substances during thermal treatment of excess sludge

The releasing characteristics of phosphorus, nitrogen compounds, organics, and some metal cations during thermal treatment of excess sludge were investigated. It was found that during heating not only phosphorus, but also nitrogen compounds, organics, and some metal cations could be released in abundance. The maximum orthophosphate （ortho-P） release of about 90 mg/L in concentration was observed at 50℃ in 1 h. Except for volatile fatty acids （VFAs）, comparatively little total nitrogen （TN）, total organic carbon （TOC）, and metal cations were released at the same time. Such results might favor further process of phosphorus recovery. VFAs were considerably released only at 50℃. Acetic, butyric, and propionic acid were the most abundant components in turn and their releasing profiles exhibited good linear relationship with time （R2 = 0.9977, 0.9624, and 0.8908, respectively）. The concentrations of Mg^2＋ and K^＋ increased with time and temperature during thermal treatment, but Ca^2＋ decreased. The release of Mg^2＋ and K^＋ agreed well with TP release （R^2 = 0.9892 and 0.9476, respectively）. Temperature in the experimental range had very little impact on the linear relationships, especially of Mg^2＋. Moreover, the parameter of mixed liquor suspended solids （MLSS） was found to be an important factor for thermal sludge treatment as the released ortho-P and total phosphorus （TP） at 50℃ increased more than one-fold when MLSS was increased from 4000 to 8000 mg/L.

Effect of Sodium Ion Concentration on Hydrogen Production from Sucrose by Anaerobic Hydrogen-producing Granular Sludge

This work evaluated the effects of sodium ion concentration, ranging from 0 to 16000mg·L^-1（Na^＋）, on the conversion of sucrose to hydrogen by a high-activity anaerobic hydrogen-producing granular sludge. At the optimum sodium ion concentration [1000-2000mg·L^-1（Na^＋）] for hydrogen production at 37℃, the maximum sucrose degradation rate, the specific hydrogen production yield and the specific hydrogen production rate were 393.6-413.1mg·L^-1.h^-1, 28.04-28.97ml·g^-1, 7.52-7.83ml·g^-1.h^-1, respectively. The specific production yields of propionate, butyrate and valerate decreased with increasing sodium ion concentration, whereas the specific acetate production yield increased, meanwhile the specific production yields of ethanol and caproate were less than 55.3 and 12.6mg·g^-1, respectively. The hybrid fermentation composition gradually developed from acetate, propionate and butyrate to acetate with the increase in sodium ion concentration.

Transformation of organic matters in fresh leachate during anaerobic degradation under long hydraulic retention time

This study investigated the biodegradability of fresh leachate from a young municipal solid waste landfill at the inoculum to substrate ratios （V/V） of 10/100, 25/100, 50/100 and 100/100, as well as the transformation of organic matters in leachate under prolonged hydraulic retention time （HRT） of 50 d. Fresh leachate showed a good biodegradability, with chemical oxygen demand （COD） removal efficiency as high as 87%-92% and cumulative methane yield close to theoretical value （0.35 L CH4/g CODdeg）. Methane production and COD depletion presented biphasic characteristics associated with the successive utilization of two major intermediates, acetate and propionate, The biphasic degradation of fresh leachate was resulted from the different hydrolysis rates of diverse substrates and the changes in microorganism community structure. After 50 d, the effluents were dominated by high-MW organic compounds （MW 〉 10 kDa） at each inoculum ratio, which might be refractory compounds released from cell lyses.

Potassium ferrate pretreatment promotes short chain fatty acids yield and antibiotics reduction in acidogenic fermentation of sewage sludge

During the acidogenic fermentation converting waste activated sludge (WAS) into shortchain fatty acids (SCFA), hydrolysis of complex organic polymers is a limiting step and the transformation of harmful substances (such as antibiotics) during acidogenic fermentation is unknown. In this study, potassium ferrate (KFeO) oxidation was used as a pretreatment strategy for WAS acidogenic fermentation to increase the hydrolysis of sludge and destruct the harmful antibiotics. Pretreatment with KFeOcan effectively increase the SCFA production during acidogenic fermentation and change the distribution of SCFA components.With the dosage of 0.2 g/g TS, the maximum SCFA yield was 4823 mg COD/L, which is 28.3times that of the control group;acetic acid accounts for more than 90% of the total SCFA. The higher dosage (0.5 g/g TS) can further increase the proportion of acetic acid, but inhibit the overall performance of SCFA production. Apart from the promotion of hydrolysis and acidogenesis, KFeOpretreatment can also simultaneously oxidizes and degrades part of the antibiotics in the sludge. When the dosage is 0.5 g/g TS, the degradation efficacy of antibiotics is the most significant, and the contents of ofloxacin, azithromycin, and tetracycline in the sludge are reduced by 69%, 42%, and 50%, respectively. In addition, KFeOpretreatment can also promote the release of antibiotics from sludge flocs, which is conducive to the simultaneous degradation of antibiotics in the subsequent biological treatment process.

Effect of Sodium Ion Concentration on Hydrogen Production from Sucrose by Anaerobic Hydrogen-producing Granular Sludge

This work evaluated the effects of sodium ion concentration, ranging from 0 to 16000mg·L-1(Na+), on the conversion of sucrose to hydrogen by a high-activity anaerobic hydrogen-producing granular sludge. At the opti- mum sodium ion concentration [1000—2000mg·L-1(Na+)] for hydrogen production at 37℃, the maximum sucrose degradation rate, the specific hydrogen production yield and the specific hydrogen production rate were 393.6— 413.1mg·L-1·h-1, 28.04—28.97ml·g-1, 7.52—7.83ml·g-1·h-1, respectively. The specific production yields of propionate, butyrate and valerate decreased with increasing sodium ion concentration, whereas the specific acetate production yield increased, meanwhile the specific production yields of ethanol and caproate were less than 55.3 and 12.6mg·g-1, respectively. The hybrid fermentation composition gradually developed from acetate, propionate and butyrate to acetate with the increase in sodium ion concentration.

Effect of Cu^(2+) concentration on hydrogen fermentation

The results of the study on the effect of Cu2+ concentration on hydrogen production using a glucose solution and mixed microorganisms from a 9 2 L laboratory-scale digester operated at 35 ℃ indicated the productions of hydrogen, volatile fatty acids （VFAs）, ethanol and biogas were inhibited by the added Cu2+ which caused a reduction in the glucose degradation efficiency. The specific yields of hydrogen, ethanol and acetate decreased with increased Cu2+ concentration in the mixed microorganisms. When the Cu2+ concentration in the mixed microorganisms was almost an constant, the specific yields of hydrogen, ethanol and acetate were very similar. A 50% inhibition of hydrogen yield (EC50) occurred at a Cu2+ dosage of 0 50 mg/L in the present investigation. Cu2+ in the external environment is accumulated in the mixed microorganisms. The Cu2+ speciation, using a sequential extraction technique, showed that the Cu2+ was present mainly bound to organic sites in the mixed microorganisms in the present investigation.