Dynamic behavior in response to pH shift during anaerobic acidogenesis with a chemostat culture

To clarify the dynamic behavior of the anaerobic acid reactor in response to pH changes, a continuous cultivation was performed. By stepwise shifting the culture pH in the acid reactor from 6.0 to 8.0, the main products were changed from butyric acid to acetic and propionic acids. This phenomenon was reproducible, reversible and was not affected by the dilution rate. It was considered that the dominant microbial populations changed in the acid reactor due to the pH shift.     

Multi-omic profiling reveals associations between the gut mucosal microbiome,the metabolome,and host DNA methylation associated gene expression in patients with colorectal cancer

Actinomyces oris is an early colonizer and has two types of fimbriae on its cell surface, type 1 fimbriae (FimP and FimQ) and type 2 fimbriae (FimA and FimB), which contribute to the attachment and coaggregation with other bacteria and the formation of biofilm on the tooth surface, respectively. Short-chain fatty acids (SCFAs) are metabolic products of oral bacteria including A. oris and regulate pH in dental plaques. To clarify the relationship between SCFAs and fimbrillins, effects of SCFAs on the initial attachment and colonization (INAC) assay using A. oris wild type and fimbriae mutants was investigated. INAC assays using A. oris MG1 strain cells were performed with SCFAs (acetic, butyric, propionic, valeric and lactic acids) or a mixture of them on human saliva-coated 6-well plates incubated in TSB with 0.25% sucrose for 1 h. The INAC was assessed by staining live and dead cells that were visualized with a confocal microscope. Among the SCFAs, acetic, butyric and propionic acids and a mixture of acetic, butyric and propionic acids induced the type 1 and type 2 fimbriae-dependent and independent INAC by live A. oris, but these cells did not interact with streptococci. The main effects might be dependent on the levels of the non-ionized acid forms of the SCFAs in acidic stress conditions. GroEL was also found to be a contributor to the FimA-independent INAC by live A. oris cells stimulated with non-ionized acid. SCFAs affect the INAC-associated activities of the A. oris fimbrillins and non-fimbrillins during ionized and non-ionized acid formations in the form of co-culturing with other bacteria in the dental plaque but not impact the interaction of A. oris with streptococci.     

Study of the acidogenic phase of the anaerobic fermentation of stillage from ethanol distilleries

Summary A bench scale continuously stirred reactor was used to study the acidogenic phase of the anaerobic fermentation of stillage. The residence time of the effluent in the reactor ranged from 15.7 to 8.2 hours, pH was around 5.0 and temperature was maintained at 35°C. The results indicate that the residence time had no appreciable effect on the production or composition of the organic acids. The main acid products found in the reactor effluent were acetic, propionic and butiric acids.     

Characterization of aerobic,facultative anaerobic,and anaerobic bacteria in an acidogenic phase reactor and their metabolite formation

Fifty-two aerobic and facultative anaerobic and 57 anaerobic bacterial isolates were obtained from an acidogenic phase digestion system. These isolates were characterized and the similarities between the different strains were calculated using Sokal and Michener's similarity coefficient. The aerobic and facultative anaerobic strains clustered in two major groups with the strains of the first main group being gram-negative fermentative rods, representing the generaKlebsiella, Enterobacter, Escherichia andAeromonas. Isolates of the second group were gram-positive streptococci similar toStreptococcus lactis. The strict anaerobic isolates also clustered into two main groups with strains of cluster A being identified as members of the genusFusobacterium while strains in cluster B were members of the genusBacteroides. Hypothetical mean organisms were calculated for each cluster and used in further culture studies. The major products of the continuously fed acidogenic phase reactor were ethanol and acetic, propionic, and butyric acids. In batch cultures, ethanol, acetic acid, diacetyl, and 2,3-butanediol were formed by the strains as major products both under aerobic and anaerobic conditions. The ability of the aerobic and facultative anaerobic strains to be metabolically active under anaerobic conditions indicates a prominent role in acidogenic reactors.     

Characterization of anaerobic microbial culture with high acidogenic activity

The mixed cultures which were used were isolated from municipal sludge digesters, and the production of organic acids (acetic, propionic, butyric, etc.) from carbohydrates was tested. The behavior of the reference population (culture R) obtained directly from the sewage treatment plant, is compared to that obtained after three months in a plug-flow reactor (Gradostat fermentor) without pH control (culture A) and after six months with pH control (culture B). For culture B, the specific rate of acid production is related to the cell growth rate by (1/X)r_p= 17 µ + 1.6 with a maximal acid concentration of 40 g/liter. The batch culture yields are improved from 0.36g/g for the initial culture (R) to 0.72 g/g for culture B after six months in continuous culture, and 0.8 g/g in plug-flow continuous culture. The productivity of organic acids reaches 1.7 g/liter·hr. It is suggested that the acidogenic fermentation, the first step of methanogenesis, is a potential process to produce acetic, propionic, and butyric acids.     

The effect of pH and temperature manipulation on metabolite composition during acidogenesis in a hybrid anaerobic digester

Summary A synthetic medium containing 9 g/l sucrose was hydrolyzed in a novel hybrid reactor. A minimum hydraulic retention time (HRT) of 9.9 h, with a gas production rate of 1.07 m³/m³·d, was obtained without continuous neutralization. A viable anaerobic cell count of 10⁹ organisms/ml was obtained in the reactor fluid. The results showed that both pH and temperature significantly influenced the type and concentration of the various metabolites formed. These include ethanol, formic, acetic, propionic and butyric acids as primary metabolites and caproic acid as secondary metabolite. From the results obtained, it is suggested that to obtain the energetically most favourable products, a substrate pH of 6.5 and a temperature of 35°C must be used in anaerobic acidogenic digesters.     

Volatile fatty acids distribution during acidogenesis of algal residues with pH control

The anaerobic acidification of protein-rich algal residues with pH control (4, 6, 8, 10) was studied in batch reactors, which was operated at mesophilic(35 °C) condition. The distribution of major volatile fatty acids (VFAs) during acidogenesis was emphasized in this paper. The results showed that the acidification efficiency and VFAs distribution in the acid reactor strongly depended on the pH. The main product for all the runs involved acetic acid except that the proportion of butyric acid acidified at pH 6 was relatively higher. The other organic acids remained at lower levels. The VFAs yield reached the maximum value with about 0.6 g VFAs/g volatile solid (VS) added as pH was 8, and also the content of total ammonia nitrogen (TAN) reached the highest values of 9,629 mg/l. Low acidification degrees were obtained under the conditions at pH 4 and 10, which was not suitable for the metabolism of acidogens. Hydralic retention time (HRT) required for different conditions varied. As a consequence, it was indicated that pH was crucial to the acidification efficiency and products distribution. The investigation of acidogenesis process, which was producing the major substrates, short-chain fatty acids, would play the primary role in the efficient operation of methanogenesis.     

Production of PHA from starchy wastewater via organic acids

Polyhydroxyalkanoate (PHA) was produced from a starchy wastewater in a two-step process of microbial acidogenesis and acid polymerization. The starchy organic waste was first digested in a thermophilic upflow anaerobic sludge blanket (UASB) reactor to form acetic (60-80%), propionic (10-30%) and butyric (5-40%) acids. The total volatile fatty acids reached 4000 mg l(-1) at a chemical oxygen demand (COD) loading rate of 25-35 g l(-1) day(-1). A carbon balance indicates that up to 43% of the organic carbon in the starchy waste went to the organic acids and the rest to biogas, volatile suspended solids and residual sludge accumulated in the reactor. The acid composition profile was affected by COD loading rate: a medium rate around 9 g l(-1) day(-1) gave a high propionic acid content (29% wt) and a high rate around 26 g l(-1) day(-1) led to a high butyric acid content (34% wt). The acids in the effluent solution after microfiltration were utilized and polymerized into PHA by bacterium Alcaligenes eutrophus in a second reactor. Fifty grams of PHA was produced from 100 g total organic carbon (TOC) utilized, a yield of 28% based on TOC, which is comparable with 55 g PHA per 100 g TOC of pure butyric and propionic acids used. PHA formation from individual acids was further investigated in a semi-batch reactor with three acid feeding rates. With a limited nitrogen source (80-100 mg NH(3) per liter), the active biomass of A. eutrophus, not including the accumulated PHA in cells, was maintained at a constant level (8-9 g l(-1)) while PHA content in the cell mass increased continuously in 45 h; 48% PHA with butyric acid and 53% PHA with propionic acid, respectively. Polyhydroxybutyrate was formed from butyric acid and poly(hydroxybutyrate-hydroxyvalerate) formed from propionic acid with 38% hydroxyvalerate.     

Assessment of toxicity of volatile fatty acids to Photobacterium phosphoreum

The toxicity of four volatile fatty acids (VFAs) as anaerobic digestion (AD) intermediates was investigated at pH 7. Photobacterium phosphoreum T3 was used as an indicator organism. Binary, ternary and mixtures of AD intermediates were designated by letters A (acetic acid + propionic acid), B (acetic acid + butyric acid), C (acetic acid + ethanol), D (propionic acid + butyric acid), E (propionic acid + ethanol), F (butyric acid + ethanol), G (acetic acid + propionic acid + butyric acid), H (acetic acid + propionic acid + ethanol), I (acetic acid + butyric acid+ ethanol), J (propionic acid + butyric acid + ethanol) and K (acetic acid + propionic acid + butyric acid + ethanol) to assess the toxicity through equitoxic mixing ratio method. The IC₅₀ values of acetic acid, propionic acid, butyric acid and ethanol were 9.812, 7.76, 6.717 and 17.33 g/L respectively, displaying toxicity order of: butyric acid > propionic acid > acetic acid > ethanol being additive in nature. The toxic effects of four VFAs could be designated as synergistic and one additive in nature.     

Anaerobic digestion: microbial and biochemical aspects of volatile acid production

Summary The production of organic acids has been tested with bacterial flora selected from a municipal sludge digestor. In order to elucidate the basic mechanisms by which glucose is converted to volatile fatty acids, the examination of non-methanogenic bacteria was attempted. Both lactate-producers and lactate-utilizers were found among these bacteria. When mixed isolates were used as the inoculum, the accumulation of lactic acid and its further conversion to propionic and butyric acids was demonstrated at a carbon conversion rate of about 0.75. It is therefore suggested that this metabolic sequence may occur as a normal process in acidogenic fermentation, which is the first step in anaerobic digestion.     

Anaerobic degradation of dehydrodiisoeugenol by rumen bacteria

The degradation of dehydrodiisoeugenol (DDIE) by cow rumen bacteria was studied under strictly anaerobic conditions. After two days of cultivation, about 23% of DDIE (1.2 mM) was degraded to volatile fatty acids (VFA) such as acetic acid, propionic acid and butyric acid. The aromatic intermediates were vanillic acid, 5-methylvanillin and 3-methyl-4-hydroxybenzaldehyde, which suggested that the coumaran ring in DDIE was cleaved during degradation. These results indicate that the rumen anaerobes can degrade this lignin-related dimer to monoaromatic compounds and VFA.     

Microbial interactions associated with secondary cucumber fermentation

Aims

To evaluate the interaction between selected yeasts and bacteria and associate their metabolic activity with secondary cucumber fermentation.

Methods and Results

Selected yeast and bacteria, isolated from cucumber secondary fermentations, were inoculated as single and mixed cultures in a cucumber juice model system. Our results confirmed that during storage of fermented cucumbers and in the presence of oxygen, spoilage yeasts are able to grow and utilize the lactic and acetic acids present in the medium, which results in increased brine pH and the chemical reduction in the environment. These conditions favour opportunistic bacteria that continue the degradation of lactic acid. Lactobacillus buchneri, Clostridium bifermentans and Enterobacter cloacae were able to produce acetic, butyric and propionic acids, respectively, when inoculated in the experimental medium at pH 4·6. Yeast and bacteria interactions favoured the survival of Cl. bifermentans and E. cloacae at the acidic pH typical of fermented cucumbers (3·2), but only E. cloacae was able to produce a secondary product.

Conclusions

The methodology used in this study confirmed that a complex microbiota is responsible for the changes observed during fermented cucumber secondary fermentation and that certain microbial interactions may be essential for the production of propionic and butyric acids.

Significance and Impact of the Study

Understanding the dynamics of the development of secondary cucumber fermentation aids in the identification of strategies to prevent its occurrence and economic losses for the pickling industry.     

Effect of Short-Chain Fatty Acids and Soil Atmosphere on Tylenchorhynchus spp.

Short-chain fatty acids can be produced under anaerobic conditions by fermentative soil microbes and have nematicidal properties. We evaluated the effects of butyric and propionic acids on death and recovery of stunt nematodes (Tylenchorhynchus spp.), a common parasite of turfgrass. Nematodes in a sand-soil mix (80:20) were treated with butyric or propionic acid and incubated under air or N₂ for 7 days at 25 °C. Amendment of soil with 0.1 and 1.0 µmol (8.8 and 88 µg) butyric acid/g soil or 1.0 µmol (74 µg) propionic acid/g soil resulted in the death of all nematodes. The composition of the soil atmosphere had no effect on the nematicidal activity of the acids. Addition of hydrochloric acid to adjust soil pH to 4.4 and 3.5 resulted in nematode mortality relative to controls (41% to 86%) but to a lesser degree than short-chain fatty acids at the same pH. Nematodes did not recover after a 28-day period following addition of 10 µmol butyric acid/g soil under air or N₂. Carbon mineralization decreased during this period, whereas levels of inorganic N and microbial biomass-N remained constant. Short-chain fatty acids appear to be effective in killing Tylenchorhynchus spp. independent of atmospheric composition. Nematode mortality appears to be a function of the type and concentration of fatty acid and soil pH.     

Cytotoxic effects of culture supernatants of oral bacteria and various organic acids on Vero cells

Dilute culture filtrates of the three asaccharolytic black-pigmented Bacteroides species as well as B. macacae, Fusobacterium nucleatum, and animal strains resembling B. gingivalis were cytotoxic for the African green monkey kidney cell line (Vero cells). Butyric or propionic acid, major metabolic products of these bacteria, seem to be responsible for the characteristic morphological changes observed. Among the organic acids studied, only butyric, propionic, and valeric acids exhibited cytotoxic effects. Finally, cumulative effects (as opposed to synergistic) were observed when mixtures of two organic acids were tested.     

Search for propionic acid bacteria in the human intestine

The possibility of detecting propionic acid-producing bacteria in the intestine of healthy humans with a view to obtaining a strain which is physiologically most suitable for therapeutic purposes has been studied. The selective conditions for the isolation of propionic acid-producing bacteria from the large intestine have been experimentally established. Analysis of 70 puncture biopsy specimens of parietal mucus and luminal contents from different sections of the intestine has not shown the presence of the representatives of propionic acid-producing bacteria. The strains isolated under the conditions selective for such bacteria have been found to belong to the family Enterobacteriaceae. These strains have proved capable of synthesizing vitamin B12, but the synthesis of propionic acid has not been observed.     

Effect of Organic Acids on Shrimp Pathogen, <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

Shrimp farming accounts for more than 40% of the world shrimp production. Luminous vibriosis is a shrimp disease that causes major economic losses in the shrimp industry as a result of massive shrimp kills due to infection. Some farms in the South Asia use antibiotics to control Vibrio harveyi, a responsible pathogen for luminous vibriosis. However, the antibiotic-resistant strain was found recently in many shrimp farms, which makes it necessary to develop alternative pathogen control methods. Short-chain fatty acids are metabolic products of organisms, and they have been used as food preservatives for a long time. Organic acids are also commonly added in feeds in animal husbandry, but not in aquaculture. In this study, growth inhibitory effects of short-chain fatty acids, namely formic acid, acetic acid, propionic acid, and butyric acid, on V. harveyi were investigated. Among four acids, formic acid showed the strongest inhibitory effect followed by acetic acid, propionic acid, and butyric acid. The minimum inhibitory concentration (MIC) of 0.035% formic acid suppressed growth of V. harveyi. The major inhibitory mechanism seems to be the pH effect of organic acids. The effective concentration 50 (EC₅₀) values at 96 h inoculation for all organic acids were determined to be 0.023, 0.041, 0.03, and 0.066% for formic, acetic, propionic, and butyric acid, respectively. The laboratory study results are encouraging to formulate shrimp feeds with organic acids to control vibrio infection in shrimp aquaculture farms.     

Characteristics of spoilage-associated secondary cucumber fermentation

Secondary fermentations during the bulk storage of fermented cucumbers can result in spoilage that causes a total loss of the fermented product, at an estimated cost of $6,000 to $15,000 per affected tank. Previous research has suggested that such fermentations are the result of microbiological utilization of lactic acid and the formation of acetic, butyric, and propionic acids. The objectives of this study were to characterize the chemical and environmental conditions associated with secondary cucumber fermentations and to isolate and characterize potential causative microorganisms. Both commercial spoilage samples and laboratory-reproduced secondary fermentations were evaluated. Potential causative agents were isolated based on morphological characteristics. Two yeasts, Pichia manshurica and Issatchenkia occidentalis, were identified and detected most commonly concomitantly with lactic acid utilization. In the presence of oxygen, yeast metabolic activities lead to lactic acid degradation, a small decline in the redox potential (E(h), Ag/AgCl, 3 M KCl) of the fermentation brines, and an increase in pH to levels at which bacteria other than the lactic acid bacteria responsible for the primary fermentation can grow and produce acetic, butyric, and propionic acids. Inhibition of these yeasts by allyl isothiocyanate (AITC) resulted in stabilization of the fermented medium, while the absence of the preservative resulted in the disappearance of lactic and acetic acids in a model system. Additionally, three Gram-positive bacteria, Lactobacillus buchneri, a Clostridium sp., and Pediococcus ethanolidurans, were identified as potentially relevant to different stages of the secondary fermentation. The unique opportunity to study commercial spoilage samples generated a better understanding of the microbiota and environmental conditions associated with secondary cucumber fermentations.     

Survival of a Salmonella typhimurium poultry isolate in the presence of propionic acid under aerobic and anaerobic conditions

Propionic acid is commonly found as a fermentation product in the gastrointestinal tracts of food animals and has also been used to limit the microbial contaminants in animal feeds. Because propionic acid is known to have antibacterial activity, the propionic acid encountered by foodborne pathogens during their life cycles may play an important role in inhibiting the survival of the pathogens. The survival patterns of Salmonella typhimurium poultry isolate were determined both in aerobic and anaerobic tryptic soy broth (TSB; pH 5.0 or 7.0) containing various concentrations of propionic acid (0-200 mM). The levels of recovered cells were consistently greater at pH 7.0 compared to those at pH 5.0. For the first 4 days, the levels were significantly decreased by incubation under anaerobic conditions as compared to aerobic condition at pH 7.0 (P<0.05). However, there were fluctuations of cell populations with different patterns depending on both concentrations and growth conditions. To characterize the nature of the capability which allowed the cell multiplication following decreases in cell population during incubation at pH 7.0, the cells isolated from the outgrowth cultures were tested for survival in aerobic or anaerobic TSB (pH 5.0 or pH 7.0) containing propionic acid (50 mM). The outgrowth isolates did not show significant differences in the level of recovered cells in the presence of propionic acid when compared to the wild type strain (P>0.05), suggesting that the cells in the outgrowth cultures did not harbour mutation(s) conferring increased resistance to propionic acid. In addition, the level of recovered cells of isogenic rpoS mutant strain of S. typhimurium was not significantly different from that of the wild type strain in the same assay conditions (P<0.05). The results of this study show that the bactericidal activity of propionic acid on S. typhimurium can be affected by environmental conditions such as acidic pH levels and anaerobiosis in food materials and gastrointestinal tracts. However, S. typhimurium is also able to multiply in the presence of sublethal concentrations of propionic acid at neutral pH during prolonged incubation under both aerobic and anaerobic conditions.     

Modeling of two-phase anaerobic process treating traditional Chinese medicine wastewater with the IWA Anaerobic Digestion Model No. 1

The aim of the study was to implement a mathematical model to simulate two-phase anaerobic digestion (TPAD) process which consisted of an anaerobic continuous stirred tank reactor (CSTR) and an upflow anaerobic sludge blanket (UASB) reactor in series treating traditional Chinese medicine (TCM) wastewater. A model was built on the basis of Anaerobic Digestion Model No. 1 (ADM1) while considering complete mixing model for the CSTR, and axial direction discrete model and mixed series connection model for the UASB. The mathematical model was implemented with the simulation software package MATLABTM/Simulinks. System performance, in terms of COD removal, volatile fatty acids (VFA) accumulation and pH fluctuation, was simulated and compared with the measured values. The simulation results indicated that the model built was able to well predict the COD removal rate (−4.8–5.0%) and pH variation (−2.9–1.4%) of the UASB reactor, while failed to simulate the CSTR performance. Comparing to the measured results, the simulated acetic acid concentration of the CSTR effluent was underpredicted with a deviation ratios of 13.8–23.2%, resulting in an underprediction of total VFA and COD concentrations despite good estimation of propionic acid, butyric acid and valeric acid. It is presumed that ethanol present in the raw wastewater was converted into acetic acid during the acidification process, which was not considered by the model. Additionally, due to the underprediction of acetic acid the pH of CSTR effluent was overestimated.     

Ethanol production from glucose byClostridium thermosaccharolyticum strains: Effect of pH and temperature

Summary The fermentation of glucose byClostridium thermosaccharolyticum strains IMG 2811^T, 6544 and 6564 was studied in batch culture in a complex medium at different temperatures in defined and free-floating pH conditions. All the strains ferment 5 g glucose.l^–1 completely. The yield of the fermentation products turned out to be independent of the incubation temperature for strain IMG 2811^T. Strain IMG 6544 produced at 60°C significantly more ethanol and less acetic acid, butyric acid, hydrogen gas and biomass than at lower temperatures. With strain IMG 6564, the opposite effect occurred: ethanol appeared to be the main fermentation product at 45°C; at 60°C less ethanol and more acetic acid, butyric acid and hydrogen gas was formed.Experiments, carried out with strain IMG 6564, at defined pH conditions (between 5.5 and 7) and different temperatures (45, 55 and 60°C) revealed no effect of the incubation temperature, but an important effect of the pH on the product formation. At pH 7, ethanol was the main fermentation product while minor amounts of hydrogen gas, acetic and butyric acid were produced. Lowering the pH gradually to 5.5 resulted in a decrease of ethanol and an increase of biomass, hydrogen gas, acetic, butyric and lactic acids. At pH higher than 7 no growth occurred. Similar conclusions could be drawn for strains IMG 2811^T and 6544.