Development of a two-phase combination fermenter for the conversion of cellulose to methane

Cellulose degradation to methane under continuous fermentation conditions was compared using fully mixed, fully mixed with solids return, sludge-blanket, and fixed-film fermenters. In fully mixed fermenters, a decrease in hydraulic retention time (HRT) of two weeks or less caused the wash out of anaerobes capable of converting volatile fatty acids to methane, while at increased feeding rates over 1 g/L day the rapid growth of cellulolytic anaerobes upset the balance between acid formation and its conversion to CH(4). Circulation of cellulose and difficulty in settling of cellulose with attached bacteria imposed problems in the use of other types of fermenters. On the basis of information obtained from this study, a fermenter which combined a fully mixed phase for cellulose degradation and a fixed-film phase with pre-immobilized bacteria for converting fatty acids to CH(4) in one vessel, was devised. Using this fermenter, a mixed culture converted cellulose to CH(4) at 4.8 g/L day at a HRT of six days as compared to 0.7 g/L day at a HRT of 28 days in the fully mixed fermenter.     

Swine manure fermentation for hydrogen production

Biohydrogen fermentation using liquid swine manure as substrate supplemented with glucose was investigated in this project. Experiments were conducted using a semi-continuously-fed fermenter (8 L in total volume and 4 L in working volume) with varying pHs from 4.7 through 5.9 under controlled temperature (35 ± 1 °C). The hydraulic retention time (HRT) tested include 16, 20, and 24 h; however, in two pH conditions (5.0 and 5.3), an additional HRT of 12 h was also tried. The experimental design combining HRT and pH provided insight on the fermenter performance in terms of hydrogen generation. The results indicated that both HRT and pH had profound influences on fermentative hydrogen productivity. A rising HRT would lead to greater variation in hydrogen concentration in the offgas and the best HRT was found to be 16 h for the fermenter in this study. The best pH value in correspondence to the highest hydrogen generation was revealed to be 5.0 among all the pHs studied. There was no obvious inhibition on hydrogen production by methanogenesis when methane content in the offgas was lower than 2%. Otherwise, an inverse linear relationship between hydrogen and methane content was observed with a correlation coefficient of 0.9699. Therefore, to increase hydrogen content in the offgas, methane production has to be limited to below 2%.     

Effect of temperature and hydraulic retention time on volatile fatty acid production based on bacterial community structure in anaerobic acidogenesis using swine wastewater

To investigate the effect of hydraulic retention time (HRT) and temperature (T) on bacterial community structure and volatile fatty acids (VFAs) production of an acidogenic process, and VFA production and changes in the bacterial community in three identical automated anaerobic continuously-stirred tank reactors were analyzed using response surface analysis (RSA) and nonmetric multidimensional scaling (NMDS). For RSA, 11 trials were conducted to find the combination of T and HRT under which VFA production was greatest; VFA production was affected more by HRT than by T. To identify the bacterial community structure in each trial, DNA from each experimental point of the RSA was analyzed using denaturating gradient gel electrophoresis (DGGE), and eight bacteria species were detected. NMDS was conducted on band intensities obtained using DGGE, and bacterial community structure was affected more by T than by HRT. Taken together, these results suggest that VFA production during acidogenesis was more dependent on the physicochemical properties of acidogens, such as their specific growth rate or contact time with of substrates, than on changes in the microbial community.     

The role of HRT and low temperature on the acid-phase anaerobic digestion of municipal and industrial wastewaters

Two identical 31 completely mixed reactors with solids recycling capabilities were used to investigate the effects of hydraulic retention time (HRT) and low temperatures on volatile fatty acid (VFA) production. One reactor was fed with a 1:1 ratio of diluted primary sludge and a starch-rich industrial wastewater, while the other was fed with diluted primary sludge alone. The VFA and soluble COD concentrations and specific production rates reached their highest values at 30 h HRT and at 25 degrees C. Further increase in HRT (at 25 degrees C) or decrease in temperature (at an HRT of 30 h) resulted in lower amounts of VFA and COD produced. All parameters related to VFA and COD production were significantly higher in the industrial-municipal reactor than in the municipal-only reactor. The VFA:COD ratios were very high, with values ranging from about 0.8 to 1.0 indicating that hydrolysis was the rate-limiting step. Degradation of proteins (measured by ammonia production) was inhibited by the starch-rich wastewater in the industrial-municipal reactor, while no evidence of inhibition was found in the municipal-only reactor. This study revealed that VFA production was feasible at low temperatures (down to 8 degrees C), particularly in the presence of the industrial waste. Ultimately, the amount VFA produced was adequate, in most cases, to support subsequent biological nutrient removal (BNR) processes.     

Effect of organic loading and retention time on dairy manure fermentation

The investigations presented and discussed herein establish an enhanced understanding on volatile fatty acid (VFA) production as a function of dairy manure fermenter organic loading (OL) and retention time (RT), first through a factorial of 64 fermentation potential (FP) batch tests, followed by analysis of a continuously operated pilot-scale fermenter. The maximum observed net FP - 0.103 mg VFA produced (as COD) (mg VS applied)⁻¹ - occurred at an OL of 40.7 g VS L⁻¹ and at a RT of 6 days. The pilot-scale fermenter exhibited an average yield of 0.09 mg VFA (as COD) synthesized (mg VS applied)⁻¹, with average effluent total VFA concentrations of 6398 mg VFA (as COD) L⁻¹. The research demonstrates that FP tests are an effective method to optimize continuously operated dairy manure fermenters, and that dairy manure fermentation can yield large quantities of organic acids at short RTs and high OL rates.     

Mesophilic anaerobic digestion in a fluidised-bed reactor of wastewater from the production of protein isolates from chickpea flour

A study of the anaerobic digestion of wastewater derived from the production of protein isolates from chickpea flour was carried out in a laboratory-scale, mesophilic (35 °C) fluidised-bed reactor with saponite as bacterial support. Soluble chemical oxygen demand (SCOD) removal efficiencies in the range of 96.8–85.2% were achieved in the reactor at organic loading rates (OLR) of between 0.58 and 2.10 g chemical oxygen demand (COD)/l per day, hydraulic retention times (HRT) of between 14.9 and 4.5 days and average feed COD concentration of 9.1 g/l. Eighty-five percent of feed COD could be removed up to OLR of 2.1 g COD/l per day. The yield coefficient of methane production was 0.34 l of methane (at STP) per gram COD removed and was virtually independent of the OLR applied. Because the buffering capacity of the experimental system was maintained at favourable levels with excess total alkalinity present at all loadings, the rate of methanogenesis was not affected by loading. Experimental data indicated that a total alkalinity in the range of 1090–2130 mg/l as CaCO₃ was sufficient to prevent the pH from decreasing to below 7.2 for OLR of up to 2.7 g COD/l per day. The volatile fatty acid (VFA) levels and the VFA/alkalinity ratio were lower than the suggested limits for digester failure (0.3–0.4) for OLR and HRT up to 2.7 g COD/l per day and 3.5 days, respectively. For a HRT of 2.8 days (OLR of 3.00 g COD/l per day) the start of acidification was observed in the reactor.     

Some properties of a protease (subtilisin BPN′) immobilized to porous glass

Subtilisin BPN′ was immobilized to porous glass via isothiocyanate coupling. The pH optimum of the enzyme was shifted to the alkaline side on binding. This effect was more pronounced with ethyl lactate than with N-tosyl arginine methyl ester (TAME). Presumably, the shift is a reflection of the negative charge on the surface of the glass. The Michaelis constant and V_max of soluble subtilisin BPN′ with TAME were two and one orders of magnitude, respectively, lower than with ethyl lactate. V_max, calculated per g of active enzyme, with TAME as the substrate was not affected by immobilization, while V_max with ethyl lactate decreased greater than tenfold. The apparent K_M decreased on immobilization with ethyl lactate as substrate and increased with TAME. Results are explained in terms of diffusional resistance and a possible attraction of ethyl lactate to the glass surface. Active site titration indicated that about 25%, of the immobilized enzyme was active.     

Production of volatile fatty acids from bagasse by rumen bacteria

Summary Conditions are described for converting bagasse lignocellulose to volatile fatty acids (VFA) by anaerobic fermentation. Although yields of VFA were as high as 74% by weight of digestible organic matter (or 54% of dry bagasse), limitations were imposed by both fermenter design and fibre digestibility. All fermentations were substrate-limited up to the maximum initial concentration examined of 50 g bagasse · l^-1 and no product inhibition was evident (up to 260 mM VFA produced). Maximum VFA productivities of 0.25 to 0.65 g · l^-1 · h^-1 were obtained in batch fermentations and this is greater than those previously reported using lignocellulosic substrates. Batch fermentations neared completion after 66 h.     

Flocculants effect in biomass retention in a UASB reactor treating dairy manure

The performance and biomass retention of an upflow anaerobic sludge bed (UASB) reactor treating liquid fraction of dairy manure has been investigated at several organic loading rates. Two identical UASB reactors were employed. The biomass of one UASB reactor (FBR) had previously been treated with a cationic polyacrylamide, the other reactor was operated as a control reactor (CR). At 3 and 2 days of HRT both reactors functioned similarly, but at 1.5 days HRT some differences were observed between both effluents. Mean COD(T) removal percentages were 83.4% and 76.5%; COD(VFA) values in effluents were 977 and 2682 mg l(-1) for the FBR and the CR respectively. The VSS initial value in both reactors was 25.66 g VSS, whereas after the experiment the quantities were 31.83 g VSS in the FBR and 23.18 g VSS in the CR reactors. Polymer addition resulted in a higher degree of biomass retention and better performance in the FBR reactor.     

Phytoremediation of soils contaminated with phenanthrene and cadmium by growing willow (Salix × Aureo-Pendula CL 'j1011')

To assess the phytoremediation potential of an autochthonous willow (Salix × aureo-pendula CL 'J1011') for phenanthrene (PHE)-contaminated soils and PHE-cadmium (PHE-Cd) co-contaminated soils, we conducted field experiments in the lower reaches of the Yangtze River, China. Ethylenediaminetetraacetic acid (EDTA) and ethyl lactate were tested for individual and combined effects on the phytoremediation efficiency. For PHE-contaminated soils, willow plus ethyl lactate resulted in significant removal of PHE from soils after 45 days, and the PHE concentration in the shoots was significantly higher with than without ethyl lactate. For PHE-Cd co-contaminated soils, both willow plus EDTA and willow plus EDTA and ethyl lactate led to a significant decrease in the concentrations of PHE and Cd in the soils after 45 days, whereas willow alone did not. The PHE and Cd concentrations in the willow shoots were significantly enhanced in the presence of EDTA alone and with ethyl lactate, except for the PHE concentration in stems with EDTA alone. Under the same treatment, the presence of Cd had no significant influence on the PHE removal from soils. The results indicate the feasibility of using this willow together with both EDTA and ethyl lactate for the simultaneous removal of PHE and Cd from soils.     

Anaerobic acidogenesis of primary sludge: the role of solids retention time

This research investigates the effect of solids retention time (SRT) on the acid-phase anaerobic digestion of primary sludge. A series of experiments were conducted using two continuous-flow 3-L units with the following configuration: a completely mixed reactor (CMR) with clarifier and solids recycle and an upflow anaerobic sludge blanket (UASB) reactor. Results show that C(2) to C(5) volatile fatty acids (VFA) were the predominant compounds formed. At a constant hydraulic retention time (HRT) of 12 h, variation in SRT from 10 to 20 days resulted in a slight increase in VFA production in both systems, but at a shorter SRT (5 days) a drastic drop in acid production was observed. In addition, the percent distribution of VFA was to some extent affected by the change in SRT. On the other hand, organic matter degradation [measured by the chemical oxygen demand (COD) specific solubilization rate or the percent volatile suspended solids (VSS) reduction] appeared to be independent of SRT, at least in the range investigated. The percent soluble COD in the form of VFA, however, increased steadily with increasing SRT, approaching the 90% level at 20 days. The remaining soluble COD in the effluent from these systems may be mainly attributed to metabolic intermediates and unused soluble substrate. (c) 1994 John Wiley & Sons, Inc.     

Stereoselective reduction of 2-substituted cyclohexanones by Saccharomyces cerevisiae

A comparative study of two modifications of enzymic reduction of ethyl N-{2-{4-[(2-oxo-cyclohexyl)methyl]phe- noxy}ethyl} carbamate (1), an insect juvenile hormone bioanalog, was performed using Saccharomyces cerevisiae in two bioreactors of different size, 250-ml shake-flask and 1-l fermenter. The two major products of this reduction were obtained in 45–49% (w/w) yields but with > 99% enantiomeric purity. Their absolute configurations were assigned as ethyl (1S,2S)-N-{2-{4-[(2-hydroxycyclohexyl)methyl]phenoxy}ethyl}carbamate (2a) and ethyl (1R,2S)-N-{2-{4-[(2-hydroxycyclohexyl)methyl]phenoxy}ethyl}carbamate (3a).     

A mathematical and empirical model of the performance of a membrane-coupled anaerobic fermentor

A mathematical model was developed to describe the performance of a membrane-coupled anaerobic fermentor (MCAF)-based process. In our experimental results, higher volatile fatty acid (VFA) recovery ratios were obtained at greater filtration ratios. The VFA recovery ratio peaked at an HRT of 12 h and a membrane filtration ratio of 0.95 at a constant SRT. Based on our simulation, the HRT and filtration ratio should be maintained at less than 1 day and above 0.9, respectively, to exceed an organic materials recovery ratio of 35% at a constant SRT of 10 days. Our empirical model, which predicts the effluent VFA concentration (C _o ), described the performance of the MCAF adequately. The model demonstrated that the outlet VFA concentration was a function of three independent parameters—HLR, input organic concentration (C _i ), and membrane filtration ratio (ϕ). Multiple regression analyses were conducted using 50 measurements of the MCAF, yielding the following relationship: C _o  = 0.278ϕ^1.13 C _i ^1.93 HLR^0.11. The correlation coefficient (R ²) was 0.90. The simulation results were consistent with the observed data; therefore, due to its simplicity, this model predicts the effluent VFA concentration of an MCAF adequately.     

Comparison of two-stage thermophilic (68 degrees C/55 degrees C) anaerobic digestion with one-stage thermophilic (55 degrees C) digestion of cattle manure

A two-stage 68 degrees C/55 degrees C anaerobic degradation process for treatment of cattle manure was studied. In batch experiments, an increase of the specific methane yield, ranging from 24% to 56%, was obtained when cattle manure and its fractions (fibers and liquid) were pretreated at 68 degrees C for periods of 36, 108, and 168 h, and subsequently digested at 55 degrees C. In a lab-scale experiment, the performance of a two-stage reactor system, consisting of a digester operating at 68 degrees C with a hydraulic retention time (HRT) of 3 days, connected to a 55 degrees C reactor with 12-day HRT, was compared with a conventional single-stage reactor running at 55 degrees C with 15-days HRT. When an organic loading of 3 g volatile solids (VS) per liter per day was applied, the two-stage setup had a 6% to 8% higher specific methane yield and a 9% more effective VS-removal than the conventional single-stage reactor. The 68 degrees C reactor generated 7% to 9% of the total amount of methane of the two-stage system and maintained a volatile fatty acids (VFA) concentration of 4.0 to 4.4 g acetate per liter. Population size and activity of aceticlastic methanogens, syntrophic bacteria, and hydrolytic/fermentative bacteria were significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. The density levels of methanogens utilizing H2/CO2 or formate were, however, in the same range for all reactors, although the degradation of these substrates was significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. Temporal temperature gradient electrophoresis profiles (TTGE) of the 68 degrees C reactor demonstrated a stable bacterial community along with a less divergent community of archaeal species.     

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.     

Continuous methane fermentation and the production of vitamin B12 in a fixed-bed reactor packed with loofah

A fixed-bed reactor with acclimated methanogens immobilized on a loofah support was studied on a laboratory scale to evaluate the system producing methane from the mixture of CO(2) and H(2) gas, with the production of vitamin B(12) as a by-product. Fermentation using CO(2)/H(2) acclimated methanogens was conducted in a jar fermentor with hydraulic retention times (HRTs) of three and six days. The performance of the reactor was mainly dependent on the HRT. With an HRT of three days, the methane production rate and the vitamin B(12) concentration in the culture broth were 6.18 l/l-reactor/h and 2.88 mg/l-culture liquid; these values were 11.96 l/l-reactor/h and 37.54 mg/l-culture liquid for an HRT of six days. A higher total cell mass of methanogens retained 42.5 g dry cell/l-culture liquid was achieved in the HRT of six days. The loofah carrier immobilized almost 95% of the methanogens, which led to a more effective bio-reaction. It was also observed that the fermentation system had a better ability to buffer pH, especially for an HRT of six days.     

Continuous cultivation of rumen microorganisms,a system with possible application to the anaerobic degradation of lignocellulosic waste materials

Summary An in vitro continuous fermentation device is described which allows the maintenance of a mixed rumen microbial population under conditions similar to those in the rumen. The differences in flow rates of solids and liquids found in the rumen were established in vitro by means of a simple filter construction. A grass-grain mixture was used as a solid growth substrate. During a test period of 65 days the artificial rumen fermenter showed stable operation with respect to ciliate numbers, fibre degradation and volatile fatty acids production. Values obtained were comparable to those found in vivo. Optimal fibre degradation and volatile fatty acids production were maintained when hydraulic retention times (HRT) ranged from 11 to 14 h. At these HRT-values ciliate numbers were maintained at about 8.5×10⁴ cells per ml. Ciliate numbers declined drastically at HRT-values above 14h. A fermenter inoculated with a small volume of rumen fluid (1:100, v/v) reached normal protozoal numbers, fibre degradation and volatile fatty acids productions after a start up period of only 8 to 10 days. The possible application of rumen microorganisms for an efficient degradation of lignocellulosic waste material in an artificial rumen digester is discussed.     

The physiology of lactate production by Lactobacillus delbreuckii in a chemostat with cell recycle

The physiology of lactate production by Lactobacillus delbreuckii NRRL B-445 in a continuous fermenter with partial cell recycle has been studied and compared with that observed in a conventional chemostat. Partial cell recycle was achieved using a hollow-fiber ultrafiltration cartridge. The biomass growth yield was reduced in the recycle fermenter while culture viability and the cellular content of polysaccharide, protein, carbon, and nitrogen remained constant, suggesting an enlarged specific rate of glucose consumption for nonanabolic (e.g., maintenance) functions. The volumetric productivity of lactate was enhanced in the recycle fermenter due to the complete utilization of glucose. The yield of lactate from biomass and the molar product ratio, lactate: ethanol plus acetate, decreased with increasing recycle ratio. Enhanced formation of ethanol and acetate occurred in the recycle fermenter although lactate remained the major product. The change in product profile was due to glucose limitation. The specific activity of lactate dehydrogenase remained constant during recycle fermentation. These physiological observations have implications for the future application of cell recycle to production processes.     

Treatment of odorous volatile fatty acids using a biotrickling filter

In this study, a novel fibrous bioreactor was developed for treating odorous compounds present in contaminated air. The first stage of this work was a preliminary study which aimed at investigating the feasibility of using the fibrous bioreactor for the removal of malodorous volatile fatty acids (VFA) that is a common odorous contaminant generated from anaerobic degradation of organic compounds. The kinetics of microbial growth and VFA degradation in the selected culture, and the performance of the submerged bioreactor at different VFA mass loadings were studied. Above 95% of VFA removal efficiencies were achieved at mass loadings up to 22.4 g/m(3)/h. In the second stage, the odour treatment process was scaled up with system design and operational considerations. A trickling biofilter with synthetic fibrous packing medium was employed. The effects of inlet VFA concentration and empty bed retention time (EBRT) on the process performance were investigated. The bioreactor was effective in removing VFA at mass loadings up to 32 g/m(3)/h, beyond which VFA started to accumulate in the recirculation liquid, indicating the biofilm was unable to degrade all of the VFA introduced. Although VFA accumulated in the liquid phase, the removal efficiency remained above 99%. This suggested that the biochemical reaction rather than gas-liquid mass transfer was the limiting step of the treatment process. In addition, the biotrickling filter was stable for long-term operation with relatively low and steady pressure drop, no clogging and degeneration of the packing material occurred during the four-month study.     

Comparative performance and microbial diversity of hyperthermophilic and thermophilic co-digestion of kitchen garbage and excess sludge

The objective of this study was to evaluate the performance characteristics of a hyperthermophilic digester system that consists of an acidogenic reactor operated at hyperthermophilic (70 degrees C) conditions in series with a methane reactor operated at mesophilic (35 degrees C), thermophilic (55 degrees C), and hyperthermophilic (65 degrees C) conditions. Lab-scale reactors were operated continuously, and were fed with co-substrates composed of artificial kitchen garbage (TS 9.8%) and excess sludge (TS 0.5%) at the volumetric ratio of 20:80. In the acidification step, COD solubilization was in the range of 22-46% at 70 degrees C, while it was 21-29% at 55 degrees C. The average protein solubilization was 44% at 70 degrees C. The double bond fatty acid removal ratio at 70 degrees C was much higher than at 55 degrees C. These results suggested that the optimal operation conditions for the acidogenic fermenter were about 3.1 days of HRT and 4 days of SRT at 70 degrees C. Methane conversion efficiency and the VS removal percentage in the methanogenic step following acidification was around 65% and 64% on average at 55 degrees C, respectively. The optimal operational conditions for this system are acidogenesis performed at 70 degrees C and methanogenesis at 55 degrees C. The key microbes determined in the hyperthermophilic acidification step were Anaerobic thermophile IC-BH at 6.4 days of HRT and Thermoanaerobacter thermohydrosulfuricus DSM 567 at 2.4 days of HRT. These results indicated that the hyperthermophilic system provides considerable advantages in treating co-substrates containing high concentrations of proteins, lipids, and nonbiodegradable solid matter.