Phosphorus release by pure cultures of Acinetobacter sp. : effect of the growth stage with cells cultivated on various carbon sources

In order to understand biological phosphorus removal mechanisms, the role of growth stage and volatile fatty acids (VFA) on phosphate release in the anaerobic stage of P removal by three Acinetobacter strains was investigated. The phosphate release in anaerobic conditions was affected by the physiological state of cells and by the carbon source used. When the experiments were made with stationary growth phase cells, the release of phosphate was higher for all three strains cultured on acetic, propionic and butyric acid. Cells showed a limit to the amount of phosphate that could be released from total phosphate accumulated. Only 5–38% of P accumulated by the log cells and 18–58% of total P accumulated by stationary cells could be released. The ratio between the amount of P released and organic substrate removed under anaerobic condition varies depending on VFA types and tested strains.     

Phosphate Release and Uptake by Pure Cultures of Acinetobacter sp.: Effect of the Volatile Fatty Acids Concentration

Phosphorus release and uptake by pure cultures of Acinetobacter strains were investigated under anaerobic and aerobic conditions respectively. Tests were performed to study the relationship between phosphorus release-storage reaction and behavior of extracellular organic substrates: acetic, propionic, and butyric acids have been used at four concentrations (50, 100, 500, and 1000 mg · L⁻¹) in the anaerobic step of biological phosphorus removal. The results obtained depend on the strain and the volatile fatty acid (VFA) used. Phosphorus released under anaerobic condition was not always related to the amount of VFA or phosphorus consumed. Phosphorus uptake (P-uptake) in the aerobic step was found to be independent of phosphorus release rates. The best phosphorus uptake rates were obtained by Acinetobacter lwoffi ATCC21130 and Acinetobacter calcoaceticus Genoespecie SUCT-5 with butyric acid as carbon source. Received: 20 May 1996 / Accepted: 8 July 1996     

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.     

嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

嗜热子囊菌利用短链有机酸生产角质酶

以嗜热子囊菌(Thermobifida fusca WSH03-11)发酵生产角质酶为模型,研究微生物利用市政污泥厌氧酸化所产短链有机酸为碳源发酵生产高附加值产品的可能。发现：(1)以丁酸、丙酸和乙酸为碳源时,有机酸和氮元素浓度分别为8.0 g/L和1.5 g/L有利于角质酶的生产;而以乳酸为碳源时,最适有机酸和氮源浓度分别为3.0 g/L和1.0 g/L;(2)改变诱导物角质的浓度,以丁酸、丙酸、乙酸和乳酸为碳源,分别比优化前提高了31.0%、13.3%、43.8%和73.2%;(3)在四种有机酸中,T. fusca WSH03-11利用乙酸的速率最快,平均比消耗速率是丙酸的1.3倍,丁酸的2.0倍及乳酸的2.2倍;以丁酸为碳源时的酶活(52.4 U/mL)是乳酸的1.7倍、乙酸的2.5倍和丙酸的3.2倍;角质酶对乳酸的得率(12.70 u/mg)分别是丁酸的1.4倍、丙酸的3.0倍和乙酸的3.8倍;(4)以混合酸为碳源生产角质酶,T. fusca WSH03-11优先利用乙酸,而对丁酸的利用受到抑制。进一步研究发现,混合酸中0.5 g/L的乙酸将导致丁酸的消耗量降低66.7%。这是首次利用混合酸作碳源发酵生产角质酶的研究报道。这一研究结果进一步确证了利用市政污泥厌氧酸化所产有机酸为碳源发酵生产高附加值产品的可行性,为以廉价碳源生产角质酶奠定了良好的基础。     

Microbiological Degradation of Malodorous Substances of Swine Waste under Aerobic Conditions

Phenol, p-cresol, and volatile fatty acids (VFA; acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids) were used as odor indicators of swine waste. Aeration of the waste allowed the indigenous microorganisms to grow and degrade these malodorous substances. The time required for degradation of these substances varied according to the waste used, and it was not necessarily related to their concentrations. Using a minimal medium which contained one of the malodorous compounds as sole carbon source, we have selected from swine waste microorganisms that can grow in the medium. The majority of these microorganisms were able to degrade the same substrate when inoculated in sterilized swine waste but with an efficiency varying from one strain to the other. None of these strains was able to degrade all malodorous substances studied. Within 6 days of incubation these selected strains degraded the following: Acinetobacter calcoaceticus, phenol and all VFA; Alcaligenes faecalis, p-cresol and all VFA; Corynebacterium glutamicum and Micrococcus sp., phenol, p-cresol, and acetic and propionic acids; Arthrobacter flavescens, all VFA. On a laboratory scale, the massive inoculation of swine waste with C. glutamicum or Micrococcus sp. accelerated degradation of the malodorous substances. However, this effect was not observed with all of the various swine wastes tested. These results suggest that an efficient deodorization process of various swine wastes could be developed at the farm level based on the aerobic indigenous microflora of each waste.     

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.     

Interconversions and production of volatile fatty acids in the sheep rumen

1. Sheep fed at a constant rate were infused intraruminally with [1-(14)C]-acetate, -propionate or -butyrate during 5hr. periods. 2. Volatile fatty acids were estimated in the rumen contents and steady-state conditions were obtained. 3. Of the butyric acid carbon 60% was in equilibrium with 20% of the acetic acid carbon, and 2-3g.atoms of carbon were interconverted/day. 4. Little interconversion took place between propionic acid, acetic acid or butyric acid. 5. The net production rates for acetic acid, propionic acid and butyric acid were 3.7, 1.0 and 0.7moles/day respectively. 6. The production of volatile fatty acids accounted for 80% of the animal's energy expenditure.     

Effect of short-chain acids on the carboxymethylcellulase activity of the ruminal bacterium<Emphasis Type="Italic">Ruminococcus albus</Emphasis>

The addition of 100-300 mmol/L of acetic, propionic, butyric or lactic acids (short-chain acids), or of acetic, propionic, and butyric acids (volatile fatty acids, VFA) mixtures increased the degradation of carboxymethyl cellulose (CMC) by R. albus (7.5 to 46 and 6 to 39 %, respectively). Differences among individual acids were observed at 300 mmol/L whereas VFA mixtures differed at 100 mmol/L. When assayed at the same concentration, CMCase activity was increased less by NaCl than by the short-chain acids, whereas ethylene glycol decreased the activity. Since osmolarity and/or ionic strength changes in the medium cannot completely account for the observed increases of carboxymethylcellulase (CMCase) activity, it is suggested that the anions of short-chain acids produce changes in the reaction media polarity that contribute to the effects observed. Alterations in the media could also bring about conformational changes in CMCase leading to increased rates of reaction and subsequent increases in CMC degradation. Finally, explanations for the observed phenomena based on the direct effect of the compounds tested on the cellulosome complex, its domains, and/or its component enzymes are proposed.     

Volatile fatty acid concentrations in the digestive tract of the West Indian manatee, Trichechus manatus

1. Digesta samples were collected from five West Indian manatees, Trichechus manatus, for volatile fatty acid (VFA) analysis. 2. Mean total VFA concentrations were low in the stomach and duodenum (18.6 and 12.3 mM/l, respectively). Mean VFA concentrations were considerably higher in the cecum and colon (220.6 and 307.3 mM/l, respectively). 3. The relative proportions of the individual VFA's shifted from predominantly acetic acid in the foregut to a mixture of acetic, butyric and propionic acids in the hindgut. 4. The VFA concentrations in the manatee are similar to those in the dugong, Dugong dugon, and the green sea turtle, Chelonia mydas. 5. The mean total VFA concentrations indicate that the cecum and colon of the manatee are sites of microbial cellulose fermentation. The contribution of VFAs to the manatees' total energy requirements could not be calculated, but it is probably considerable.     

Removal of volatile fatty acids with immobilized Rhodococcus sp. B261

The removal of aqueous volatile fatty acids (VFA) in wastewater and spoiled waste-foods by immobilized Rhodococcus sp. B261 was investigated. The n-valeric acid (0.5%) was completely removed within 25 h under the following conditions; solution pH, 8.0; air flow rate, 0.2 l/min; superficial velocity, 0.96 h(-1); temperature, 37 degrees C. Under the optimized conditions, the acetic (8525 ppm), propionic (7310 ppm) and n-butyric (4360 ppm) except n-valeric (2572 ppm) acids from the wastewater were completely removed by immobilized Rhodococcus sp. B261 in 24 h. The acetic (7810 ppm), propionic (8942 ppm) and butyric (5730 ppm) acids from the solution of spoiled waste-foods were effectively removed by immobilized Rhodococcus sp. B261 from 48 h within 60 h but n-valeric acid (3625 ppm) took 72 h.     

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.     

Effect of 2, 4-D analogues on the induction and maintenance of callus in maize tissue culture1

Six analogues of 2, 4-D: 2(2-methyl-4-chlorophenoxy) propionic, 2(2,4-dichloro-phenoxy) propionic, 2(2-methyl-4-chlorophenoxy) butyric, 2(2,4-dichlorophenoxy) butyric, 2(2,4-dichlorophenoxy) butyric acids and the separated enantiomers of 2(2, 4, 5-trichlorophenoxy) propionic acid were examined for their ability to induce callus development and maintain its growth in maize (Zea mays) tissue cultures. The results indicate that the analogues were more effective than 2, 4-D in both respects and that alkyl substitution on the carbon side chain of the acids increased the auxin effect. It was also shown that only the (+) isomer of the two enantiomers studied, had auxin activity.     

Rates of Production of Individual Volatile Fatty Acids in the Rumen of Lactating Cows

The rumen fermentation rates in individual lactating cows were measured in four different experiments. The results disclosed that the amounts and proportions of volatile acids formed could vary widely. In one case, a marked difference in the proportions of the acids produced arose within the experiment and correlated with a difference in the proportion of methane formed.

The average rate of production per day was 10.5 moles butyric acid, 12.8 moles propionic acid, and 40 moles acetic acid. Manometric estimations of rate gave lower results than those obtained by the zero-time method, due to delay after sampling and to failure of the acids to liberate stoichiometric quantities of carbon dioxide.

For those experiments in which zero-time rates were estimated, the average specific absorption rates, i.e., the amount absorbed per hour per micromole of acid in the rumen, were 0.37 for butyric acid, 0.38 for propionic acid, and 0.26 for acetic acid.

The carbon dioxide, acids, and microbial cells produced in the rumen fermentation are estimated to account for about 90% of the carbon found in the milk and respiratory CO₂ of the cows. The carbon dioxide from the fermentation was about 27% of the carbon dioxide exhaled.

     

In vitro simulation of rabbit cecal fermentation in a semi- continuous flow fermentor. I. Role of food substrate pretreatment]

A Rusitec semi-continuous flow fermentor was used to study the influence of enzyme pretreatment of food substrates on the fermentation profile over a 2-week period following inoculation with rabbit caecal contents. Three types of substrate were examined: 1) homogenized commercial rabbit feed; 2) the solid remains of this feed after digestion with alpha-amylase for 24 h; and 3) substrate 2 digested for 4 h with pepsin (double enzyme treatment). One of a pair of nylon pouches containing 15 g substrate was replaced each day, thus producing a uniform 48-h fermentation. Fermentation of the untreated feed (1) for 5-6 days produced a fermentation profile quite different from that obtained in vivo in the rabbit caecum: propionic acid accounted for over 35% of total volatile fatty acid (VFA), and butyric acid for about 15%. Amylase digestion (2) gave a stable ferment profile closer to the in vivo profile, except that propionic and butyric acids were similar at 15% of total VFA. Digestion with both amylase and pepsin (3) produced a stable fermentation profile very close to the in vivo profile: C2 > 60%, C3 < 11% and 17% < C4 < 21%. The rate at which membrane constituents (acid detergent fibre, ADF) were lost in 48 h was similar to the digestibility coefficient measured in vivo by others for the same basic feed. Lastly, there was a high percentage (about 5%) of volatile C5 fatty acids; this could be due to the discontinuous fermentor input of one pouch per 24 h. Thus, feed pretreated with both amylase and pepsin simulates, in vitro, rabbit caecal fermentation in a semi-continuous Rusitec type fermentor.     

Microbial production of volatile fatty acids: current status and future perspectives

Volatile fatty acids (VFAs) are used as building blocks to synthesize a wide range of commercially-important chemicals. Microbially produced VFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, and isovaleric acid) can be considered as a replacement for petroleum-based VFAs due to their renewability, degradability, and sustainability. The main objective of this review is to summarize research and development of VFA production methods via microbial routes, their downstream processes, current applications, and main challenges. Various fermentation processes have been developed to produce of VFAs starting from commercially-available sugars and other raw materials such as lignocellulose, whey, and waste sludge. Only few microbes have been explored for their potential to produce VFAs, and very little genomic information data is available at the present time. There is a need to use metabolic engineering, systematic biology, evolutionary engineering, and bioinformatics to discover VFA biosynthesis routes since the pathways for isobutyric acid and isovaleric acids are still not well understood.     

Effect in the cow of intraruminal infusions of volatile fatty acids and of lactic acid on the secretion of the component fatty acids of the milk fat and on the composition of blood

1. The effects in the cow of intraruminal infusions of acetic acid, propionic acid or butyric acid on the secretion of the component fatty acids of the milk fat, and of these acids and of lactic acid on the composition of the blood plasma of the jugular vein, have been studied. 2. The infusion of acetic acid or butyric acid increased the yield of the C₄–C₁₆ acids of milk fat but decreased the yield of C₁₈ acids. The infusion of propionic acid decreased the yields of all major component acids except palmitic acid and possibly lauric acid. 3. The changes in the concentrations in blood plasma of glucose and of ketone bodies were consistent with the glucogenic effect of propionic acid and the ketogenic effects of butyric acid and acetic acid. The effects of lactic acid were not consistent from cow to cow. Only with the infusion of acetic acid was a significant increase in the concentration of total volatile fatty acids in blood plasma found. Infusions of butyric acid and of propionic acid tended to depress the concentration of citric acid in the blood plasma and infusion of acetic acid increased it. No consistent effects of the infused acids on the concentration in blood plasma of esterified cholesterol, free cholesterol, triglyceride or phospholipid were observed. 4. The possibility is discussed that the effects of the infused acids on milk-fat secretion are caused through an alteration of the concentrations of precursors of milk fat in mammary arterial blood.     

Nutrition of tempe moulds

The nutritional requirements in vitro of Amylomyces rouxii NRRL 3160, Rhizopus arrhizus NRRL 1469, R. arrhizus NRRL 1526, R. oligosporus NRRL 2710, R. oligosporus NRRL 5905, R. oligosporus NRRL A-10457, R. oryzae IMI 215407, R. oryzae NRRL 3563 and R. stolonifera NRRL A-2293 were investigated. All strains grew in glucose-ammonium-salts medium without addition of vitamins. None utilized, as sole carbon and energy source, raffinose or stachyose, the main flatulence-associated oligosaccharides in soybean. All the strains, except Rhizopus oryzae IMI 215407, utilized one or more long chain fatty acids as sole sources of carbon and energy but only the R. arrhizus strains, R. oryzae NRRL 3563 and R. stolonifera , were able to use glycerol as sole carbon and energy source. Phytic acid was used neither as a source of carbon nor of phosphate by any strain.     

Growth and pectate-lyase activity of the ruminal bacterium Lachnospira multiparus in the presence of short-chain organic acids

AIMS: Acetic, propionic, butyric and lactic acids are end products of feed fermentation by rumen microbes. The effects of these short chain acids on growth and pectate-lyase (PL) activity of Lachnospira multiparus were studied. METHODS AND RESULTS: The bacterial strain used was L. multiparus D32. Acids were tested between 50 and 300 mmol l(-1). Growth and PL activity were measured by the increase in total protein content and by the increase in absorbance at 235 nm in the reaction medium respectively. With the exception of lactic acid, all acids decreased bacterial growth rates; generally, these effects were more pronounced at higher concentrations and with acids of longer chains. PL activity was inhibited by all the acids except by butyric acid at 50 and 100 mmol l(-1). Enzyme inhibition increased with the concentrations of the acids and lactic acid was the most inhibitory. CONCLUSIONS: High concentrations of short chain acids can differentially inhibit the growth rate and the PL activity of L. multiparus. SIGNIFICANCE AND IMPACT OF THE STUDY: Products of fermentation generated by the ruminal microbiota could modify the degradation of pectic substances by this bacterium.     

Rates of fermentative digestion in the howler monkey, Alouatta palliata (primates: ceboidea)

1. Caecal material of wild howler monkeys was analyzed by gas chromatography for evidence of fermentation activity and rates of production and absorption of volatile fatty acids. 2. Results showed a high rate of production of acetic acid and lesser production of propionic, butyric and isobutyric acids. The VFA content of the blood was increase in passage through the caecal vascular system. 3. We estimate that howler monkeys may obtain as much as 31% of their required daily energy from fermentation end products. 4. Energy rich fatty acids may be of particular importance to howlers when they are living on diets high in leaves, which have high cell wall contents and low contents of nonstructural carbohydrates.