Inactivation of Salmonella typhi by high levels of volatile fatty acids during anaerobic digestion

Survival of Salmonella typhi was investigated in an anaerobic digester for cattle dung with volatile fatty acid (VFA) levels of 5000 mg l⁻¹ and pH 6·0. The organism was added to the digester only once in the first experiment and daily in the other. Survival was monitored on alternate days. In the single dose experiment, the counts of Salm. typhi declined rapidly and the pathogen was completely eliminated within 12 d in the experimental digester (VFA ca 5000 mg l⁻¹ and pH 6·0), whereas 26 d were required in the control digester (VFA ca 100 mg l⁻¹ and pH 6·8). T ₉₀ values for the experimental and control digesters were 2·44 d and 4·80 d, respectively. In the daily dose experiment, a four log reduction in the pathogen count was observed in the experimental digester, but only a two log reduction in the control digester at the end of the experimental period. The mean T ₉₀ values for the experimental and the control digester were 4·22 d and 18·63 d, respectively. In both the experiments, statistical analysis of the data showed significant differences in the survival pattern of Salm. typhi in the two digesters.     

Inactivation of Salmonella during anaerobic digestion of sewage sludge

The inactivation of Salmonella duesseldorf in sewage sludge during anaerobic digestion was investigated at 35 and 48°C with mean retention periods of between 10 and 20 days. Digesters were fed daily with raw sludge containing added Salm. duesseldorf after removal of digested sludge. During steady operation, the levels of Salm. duesseldorf in the digested and the feed sludge were determined and their specific rates of decay were estimated. The latter were: (i) greater at 48°C than at 35°C for the same retention time; (ii) similar for retention periods greater than 15 d, but lower for 10 d; (iii) greater when the level of salmonellas in the feed was lower. Gas production, a measure of steady state, was gradually lost when the mean retention period was reduced to 6.7 d. In experiments in which a single dose of Salm. duesseldorf was added to digesting sludge, the inactivation appeared to follow first-order kinetics at 35°C and the decimal decay rate, 1.6/d, was similar to that in the daily feeding experiments (1.4/d) with larger and similar inocula of Salm. duesseldorf. At 48°C, however, the rate of inactivation declined with decreasing time from inoculation suggesting that the culture contained cells differing in thermal resistance. The degrees and rates of inactivation of salmonellas in those experiments were greater than in full-scale digesters, because the latter seldom operated under conditions ideal for inactivation or because indigenous salmonellas are more resistant.     

Effect of volatile fatty acids on Shigella dysenteriae during anaerobic digestion of human night soil

Thein vitro toxic effect of different volatile fatty acids (VFA) on Shigella dysenteriae was studied in pure culture. Volatile fatty acids viz., acetate, propionate, butyrate, valerate, caproate and heptanoate, exerted pH dependent toxic effect on the pathogen, with minimum inhibitory concentration in the range of 10–3000 mg l⁻¹. The effect of high levels of VFA on S. dysenteriae was studied during anaerobic digestion of human night soil in an experimental digester with VFA level ≅ 9000 mg l⁻¹ and pH ≅ 6.5. Another digester, with VFA level ≅ 700 mg l⁻¹ and pH 7.4, served as the control. In the experimental digester, S. dysenteriae was completely eliminated within 18 days. In the control digester, a four-log reduction in pathogen count was achieved however the pathogen was not completely eliminated. T ₉₀ values for the experimental and control digesters were 2.2 and 3.7 days respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Impact of concentration, temperature, and pH on inactivation of Salmonella spp. by volatile fatty acids in anaerobic digestion

It is known that the presence of volatile fatty acids may play a role in the inactivation of pathogens for systems that employ an acid phase reactor. This study was conducted to investigate the influence of volatile fatty acids on the inactivation of Salmonella spp. over a range of digestion temperatures. In this study, digesters that were treating municipal wastewater treatment plant sludges were operated at temperatures that ranged from 35 to 49 degrees C and had a solids residence time of 15 days. Samples collected from the effluent of the digesters were dosed with solutions containing acetic, propionic, and butyric acids alone and in mixtures, and the dosed effluents were analyzed for Salmonella spp. over time. In the first round of testing, the digester effluents were dosed with individual organic acids and also a mixture containing all three volatile fatty acids over a range of concentrations from 750 to 6000 mg/L, and the pH of the samples was fixed at a value of 5.5. In the second round of testing, the sample sludges were spiked with a fixed amount of organic acid mixture, and the pH was varied from 4.5 to 7.5. The reduction of Salmonella spp. in digester effluents, when dosed with volatile organic acids, was found to depend on pH, temperature, the chain length of the acids, and the concentration and composition of the acids present. Increases in temperature appeared to increase the inhibitory effects of the volatile organic acids. At mesophilic temperatures, acidic pHs resulted in a greater inhibition of Salmonella spp.; whereas at higher temperatures neutral pHs were found to be more inhibitory. The results suggest that acid phase digesters that operate at elevated temperatures and low pH can achieve substantial reduction of Salmonella spp.     

Production of branched-chain volatile fatty acids by certain anaerobic bacteria

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.     

Production of branched-chain volatile fatty acids by certain anaerobic bacteria.

Net production of isobutyric acid, isovaleric acid, and 2-methylbutyric acid by cultures of Bacteroides ruminicola and Megasphaera elsdenii on media that contained Trypticase or casein hydrolysate continued (up to 5 days) after growth had ceased. Only trace quantities of these acids were produced in a medium that contained a mixture of amino acids that did not include the branched-chain amino acids. M. elsdenii produced increased quantities of the branched-chain fatty acids in a medium that contained Trypticase when glucose was reduced or eliminated from the culture medium. However, B. ruminicola produced increased quantities of branched-chain fatty acids and of phenylacetic acid from Trypticase when glucose was supplied at 3 mg/ml rather than at 1 mg/ml. Single strains of Streptococcus bovis, Selenomonas ruminantium, Bacteroides amylophilus, and Butyrivibrio fibrisolvens did not produce branched-chain fatty acids.     

Relative importance of trophic group concentrations during anaerobic degradation of volatile fatty acids

Although obligate syntrophic reactions cannot proceed without hydrogenotrophs, it has been unclear from the literature whether potential improvements are achievable with higher concentrations of hydrogenotrophs. In this study, the relative importance of formate-/H(2)-utilizing and acetate-utilizing trophic groups in the anaerobic degradation of butyrate and propionate was assessed by adding various proportions of these enriched cultures to a mixed anaerobic seed inoculum. The improvement resulting from the additional acetate-utilizing cultures was much greater than with formate/H(2) utilizers. Furthermore, formate/H(2) utilizers did not improve propionate utilization significantly, suggesting the importance of optimum utilization of hydrogenotrophic capacity. During most of the volatile fatty acid (VFA) degradation period, the system responded with characteristic hydrogen levels to maintain the Gibbs free energy of oxidation approximately constant for both butyrate (-6 kJ) and propionate (-14 kJ). These free-energy values were independent of methanogenic activity, as well as the volume of the seed inoculum and the VFA concentrations present. By comparing the experimental results with kinetic and mass transfer models, it was postulated that the diffusional transfer of reducing equivalents was the major limiting factor for efficient VFA degradation. Therefore, for optimum utilization of the hydrogenotrophs, low acetate concentrations are vital to enable the system to respond with higher formate/H(2) levels, thus leading to improved transfer of reducing equivalents. Due to the small number of propionate utilizers (and hence their limited surface area) and low bulk liquid concentrations, the additional formate/H(2) utilizers were of minimal use for improving the degradation rate further. The butyrate degradation rates strongly correlated with the cumulative activity of hydrogenotrophs and acetotrophs over the experimental range studied, indicating the need to model obligate syntrophic reactions as a dependent function of methanogenic activity.     

Mechanism of inhibition caused by long-chain fatty acids in anaerobic digestion process

The inhibitory effect of long-chain fatty acids on the anaerobic digestion process was examined in batch experiments using synthetic substrates. The addition of long-chain fatty acids caused the appearance of the appearance of the lag period in the methane production from acetate and in the degradation of both long-chain fatty acids and n-butyrate. Methane production from hydrogen proceeded without lag period although its rate was lowered. Fermentation of glucose was not inhibited. Neutral fat in the whole milk was easily hydrolyzed to long-chain fatty acids, which brought about the inhibition. The addition of calcium chloride reduced the inhibitory effect of long-chain fatty but it did not do so after the culture had been exposed to long-chain fatty acids for more than several hours. The addition of calcium carbonate could not reduce the inhibition because of its insolubility.     

Effects of free long-chain fatty acids on thermophilic anaerobic digestion

Summary Low concentrations of the long-chain fatty acids oleate and stearate inhibited all steps of the anaerobic thermophilic biogas process during digestion of cattle manure. The lag phase increased when the concentrations of oleate and stearate were 0.2 g/l and 0.5 g/l, respectively, and no growth was found at concentrations of 0.5 g/l for oleate and 1.0 g/l for stearate. The toxic effect of these acids was permanent as growth did not occur when inhibited cultures were diluted to a non-inhibitory concentration. No adaptation to the fatty acids toxicity was observed by pre-exposing the cultures to non-inhibitory concentrations and the inhibitory response was the same as for cultures not pre-exposed to the fatty acids. Oleate was less inhibitory when added as a neutral oil in the form of the glycerol ester. This indicates that it is the free fatty acid that influences the bacterial activity. Correspondence to: B. K. Ahring     

Hydrogen and volatile fatty acids production from marine macroalgae by anaerobic fermentation

Hydrogen and volatile fatty acids (VFAs) were coproduced from marine macroalgae by anaerobic fermentation using a microbial community. The hydrogen and VFAs production were characterized based on inoculum heat-treatment, methanogen inhibitor addition, operating temperature, and in-situ extraction of VFAs. Maximum hydrogen of 179 mL/g-VS and VFAs concentration of 9.8 g/L were produced from 35 g/L of S. japonica within 5 days of anaerobic fermentation. Hydrogen and VFAs yields were well-correlated with carbohydrate content of substrate. Inoculum heat-treatment significantly improved hydrogen production while the VFAs productivity was affected adversely. The addition of methanogen inhibitors also enhanced the hydrogen production, but the effect on VFAs production was dependent on the type of inhibitor used. Low temperature (25°C) was found to be favorable for high hydrogen and VFAs yield, while high temperature (40°C) and programmed-temperature (35 ~ 45°C) were more favorable for hydrogen and VFAs productivity. Clostridium sp. content was found to be the most abundant at 25°C. An extractive fermentation with anion-exchange resin was tested to recover the VFAs and to control the pH during the anaerobic fermentation.     

Utilization of volatile fatty acids from the anaerobic digestion liquor of sewage sludge for 5-aminolevulinic acid production by photosynthetic bacteria

Using volatile fatty acids (VFA) from the anaerobic digestion liquor of sewage sludge, up to 9.2 mm 5-aminolevulinic acid (ALA) could be produced by Rhodobacter sphaeroides under anaerobic-light (5 kLux) conditions with repeated addition of levulinic acid (LA) and glycine and using a large inoculum (approx. 2 g/l of cells, initially from glutamate/malate medium). As the VFA medium also contained organic nitrogen sources such as glutamic acid, the cells were later grown up to about 2 g/l in the VFA medium instead of the glutamate/malate medium. ALA production was then again promoted by adding LA and glycine. Using this improved method, up to 9.3 mm ALA was produced by feeding propionate and acetate together with LA and glycine, indicating that VFA medium formed from sewage sludge could be useful for ALA production.     

Effect of digestion temperature and pH on treatment efficiency and evolution of volatile fatty acids during thermophilic aerobic digestion of model high strength agricultural waste

Thermophilic aerobic digestion (TAD) of a model agricultural waste, potato peel slurry, at soluble chemical oxygen demand (COD) load equivalent to approximately 8.0 gl(-1), was carried out under batch conditions at 0.5 vvm aeration rate. Digestions were carried out at temperatures of 45, 50, 55, 60 and 65 degrees C (or left unregulated) without pH control to study the effect of digestion temperatures on TAD. The effects of digestion pH on the process were studied at pH 6.0, 7.0, 8.0, 9.0 and 9.5 (and in unregulated control) all at 55 degrees C. Except for digestion at 65 degrees C, which was inoculated extraneously using culture of Bacillus strearothermophilus all reactions were carried out using the populations indigenous to the waste. During digestion at different temperatures, the removal of soluble COD increased with temperature to reach a peak at 60 degrees C before declining slightly, removal of soluble solid (SS) followed similar pattern and reached peak at 65 degrees C being the highest temperature studied, while the degradation of TSS and TS (TSS + TS) decreased with an increase in temperature. Digestion at pH 7.0 was more efficient than at other pH values. Acetate was the predominant volatile fatty acid (VFA) in all the reactions and accounted for up to 90% of the total. Digestion at 60 degrees C led to the greatest accumulation of acetate, and this coincided with the period of highest oxygen uptake, and rapid consumption of soluble carbohydrate. Iso-valerate was also produced at all pH values. Digestion at 55 degrees C and also at pH 7.0 led to rapid and efficient processes with least accumulation of VFA and should be of interest in full-scale processes whenever it is practicable to regulate the digestion pH and temperature. The result of digestion at unregulated pH indicates that gradual adaptation may be used to achieve efficient treatment at elevated pH values. This would be of interest in full-scale processes where it is not practicable to tightly regulate digestion pH, and where the waste is produced at a pH value much higher than neutral.     

Metal-induced inhibition of anaerobic metabolism of volatile fatty acids and hydrogen

The effects of copper (Cu), chromium (Cr), cadmium (Cd), lead (Pb) and zinc (Zn) on the biotransformation of organic acids (acetate, propionate and butyrate) and H₂ were assessed in serum-bottle microcosms. Experiments were performed over a range of metal concentrations (20–200 mg/1) using biomass from an anaerobic bioreactor fed continuously with ethanol distillery waste as inoculum. In general, the added metals inhibited the biotransformation of organic acids with increasing metal concentration. However, the extent of inhibition varied for the different biotransformations and for the different metals tested. For example, the concentration of CuCl₂ effecting a 50% reduction in the rate constant for biotransformation of acetate, propionate and butyrate was 60, 75 and 30 mg/1, respectively. Cu and Cr (VI) were the most inhibitory metals in organic acid transformation, whereas Pb was the least toxic. The rate of biotransformation of acetate was reduced by half at Cu and Cr concentrations of 60 and 40 gm/1 respectively, whereas Cd, Pb, and Zn concentrations of 160 to 200 mg/l had little effect. The activities of hydrogenotrophic methanogens were much less affected by the same metals and metal concentrations.     

Medium chain length polyhydroxyalkanoate (mcl-PHA) production from volatile fatty acids derived from the anaerobic digestion of grass

A two step biological process for the conversion of grass biomass to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) was achieved through the use of anaerobic and aerobic microbial processes. Anaerobic digestion (mixed culture) of ensiled grass was achieved with a recirculated leach bed bioreactor resulting in the production of a leachate, containing 15.3 g/l of volatile fatty acids (VFAs) ranging from acetic to valeric acid with butyric acid predominating (12.8 g/l). The VFA mixture was concentrated to 732.5 g/l with a 93.3 % yield of butyric acid (643.9 g/l). Three individual Pseudomonas putida strains, KT2440, CA-3 and GO16 (single pure cultures), differed in their ability to grow and accumulate PHA from VFAs. P. putida CA-3 achieved the highest biomass and PHA on average with individual fatty acids, exhibited the greatest tolerance to higher concentrations of butyric acid (up to 40 mM) compared to the other strains and exhibited a maximum growth rate (μ_MAX?=?0.45 h^?1). Based on these observations P. putida CA-3 was chosen as the test strain with the concentrated VFA mixture derived from the AD leachate. P. putida CA-3 achieved 1.56 g of biomass/l and accumulated 39 % of the cell dry weight as PHA (nitrogen limitation) in shake flasks. The PHA was composed predominantly of 3-hydroxydecanoic acid (>65 mol%).     

Dynamics of the anaerobic process: effects of volatile fatty acids

A complex and fast dynamic response of the anaerobic biogas system was observed when the system was subjected to pulses of volatile fatty acids (VFAs). It was shown that a pulse of specific VFAs into a well-functioning continuous stirred tank reactor (CSTR) system operating on cow manure affected both CH(4) yield, pH, and gas production and that a unique reaction pattern was seen for the higher VFAs as a result of these pulses. In this study, two thermophilic laboratory reactors were equipped with a novel VFA-sensor for monitoring specific VFAs online. Pulses of VFAs were shown to have a positive effect on process yield and the levels of all VFA were shown to stabilize at a lower level after the biomass had been subjected to several pulses. The response to pulses of propionate or acetate was different from the response to butyrate, iso-butyrate, valerate, or iso-valerate. High concentrations of propionate affected the degradation of all VFAs, while a pulse of acetate affected primarily the degradation of iso-valerate or 2-methylbutyrate. Pulses of n-butyrate, iso-butyrate, and iso-valerate yielded only acetate, while degradation of n-valerate gave both propionate and acetate. Product sensitivity or inhibition was shown for the degradation of all VFAs tested. Based on the results, it was concluded that measurements of all specific VFAs are important for control purposes and increase and decrease in a specific VFA should always be evaluated in close relationship to the conversion of other VFAs and the history of the reactor process. It should be pointed out that the observed dynamics of VFA responses were based on hourly measurements, meaning that the response duration was much lower than the hydraulic retention time, which exceeds several days in anaerobic CSTR systems.     

Protein degradation in anaerobic digestion: influence of volatile fatty acids and carbohydrates on hydrolysis and acidogenic fermentation of gelatin

Summary The hydrolysis and fermentation of gelatin in the presence of a carbohydrate by gelatin-adapted mixed anaerobic bacterial populations in putatively carbon-limited chemostat cultures is investigated. It was shown that the degradation of the protein is progressively retarded with increasing dilution rates, as well as with increased concentrations of carbohydrates present in the feed as a second substrate. That this is not due to high concentrations of fermentation products in the reactor was established. Moreover, the carbohydrate is totally fermented at all dilution rates. It is concluded that for optimal performance of an anaerobic digestion system purifying waste waters containing carbohydrate/protein mixtures, fermentation of carbohydrates should be spatially separated from hydrolysis and fermentation of the protein.