Propionic Acid Degradation Research Articles

Enhanced propionic acid degradation (EPAD) system: Proof of principle and feasibility

Full-scale anaerobic single-phase digesters can be confronted with process instabilities, which often result in the accumulation of propionic acid (HPr). As a solution, an enhanced propionic acid degradation (EPAD) system has been conceptually designed and experimentally tested at lab-scale. The system consisted of two components: a liquid/solid separator containing a microfiltration membrane and an up-flow anaerobic sludge bed (UASB) reactor specialized in HPr degradation. Two lab-scale continuous stirred tank reactors (CSTR) were used, i.e. the CSTR control and the CSTR treatment. Firstly, the CSTRs were stressed by organic overloading to obtain high HPr levels. During the recovery period, besides stop feeding, no actions were taken to decrease the residual HPr concentration in the CSTR control, while the CSTR treatment was connected to EPAD system in order to accelerate its recovery. By the end of the experiment, the CSTR treatment completely recovered from HPr accumulation, while no significant decrease of the HPr level in the CSTR control was observed. Based on the experimental results, the up-scaling of EPAD system was evaluated and it would only account for about 2% of the volume of the full-scale digester, thus suggesting that the implementation of a mobile EPAD system in full-scale practice should be feasible.

Propionic acid accumulation and degradation during restart of a full-scale anaerobic biowaste digester

The methane formation rate of 300 m 3 of sludge from a full scale biowaste reactor, that was stored without feeding for six weeks during a maintenance period, was about 60% of the methanogenic activity before maintenance. The 300 m 3 sludge was then pumped back into the biowaste reactor. On the third day, after refilling of the stored biowaste suspension, anaerobic conditions were obtained and feeding was started by addition of 36.1 m 3 of fresh biowaste suspension (=11.3 tons biowaste). The pH dropped from originally pH 7.7 to pH 7.3 and later on to pH 6.8, which was considered the minimum allowed pH for methanogenesis to recover. Maximum concentrations of acetate (1.78 g l −1), n-butyrate (0.57 g l −1) and n-valerate (0.44 g l −1) accumulated during the following days with feeding of 11.8 tons on day 5 and twice 6.5 tons on days 7 and 9, respectively. Thereafter, acetate, n-butyrate and n-valerate were degraded completely, whereas the concentration of propionate was still increasing. Propionic acid was the dominant fatty acid during the restart period and reached its maximum concentration of 6.2 g l −1 17 days after start of feeding. This high level of propionate was degraded completely in about 5 days with maximum degradation rates of 2.14 g l −1 d −1, and the pH of the anaerobic sludge increased from 7.1 to 7.4. During restart, the methane content of the biogas increased successively to 65%. Samples that were taken at different time intervals during the restart phase of the methane reactor showed different fatty acid degradation capabilities. After 10 days, when acetate and n-butyrate still accumulated in the methane reactor the maximum acetate degradation rate was 1.52 g l −1 d −1 and the n-butyrate degradation rate was 0.51 g l −1 d −1. Oxidation of n-valerate caused an increase of propionate, which was degraded after a lag phase of 6 days with a maximum rate of 0.6 g l −1 d −1. In the samples taken after 16 and 23 days, the propionate degradation rate increased to 1.42 g l −1 d −1 and 1.55 g l −1 d −1, respectively, and the lag phase for propionate degradation was reduced or had disappeared completely. The maximum propionate degradation rate was measured in the methane reactor in the fourth week after restart. The synthrophic propionate oxidizing bacteria were apparently the most suffering bacteria during sludge storage. If the propionate oxidizing bacteria could be kept active and the propionate degrading activity of the biowaste suspension of 6.16 g l −1 d −1 before the maintenance period could be maintained, then accumulation of 6.2 g l −1 propionate in the methane reactor after restart could be avoided and full activity reached even earlier.

Optimised anaerobic treatment of house-sorted biodegradable waste and slaughterhouse waste in a high loaded half technical scale digester

Anaerobic co-digestion of organic wastes from households, slaughterhouses and meat processing industries was optimised in a half technical scale plant. The plant was operated for 130 days using two different substrates under organic loading rates of 10 and 12 kgCOD.m(-3).d(-1). Since the substrates were rich in fat and protein components (TKN: 12 g.kg(-1) the treatment was challenging. The process was monitored on-line and in the laboratory. It was demonstrated that an intensive and stable co-digestion of partly hydrolysed organic waste and protein rich slaughterhouse waste can be achieved in the balance of inconsistent pH and buffering NH4-N. In the first experimental period the reduction of the substrate COD was almost complete in an overall stable process (COD reduction >82%). In the second period methane productivity increased, but certain intermediate products accumulated constantly. Process design options for a second digestion phase for advanced degradation were investigated. Potential causes for slow and reduced propionic and valeric acid degradation were assessed. Recommendations for full-scale process implementation can be made from the experimental results reported. The highly loaded and stable codigestion of these substrates may be a good technical and economic treatment alternative.

Regulation of the mhp Cluster Responsible for 3-(3-Hydroxyphenyl)propionic Acid Degradation in Escherichia coli

The mhp gene cluster from Escherichia coli constitutes a model system to study bacterial degradation of 3-(3-hydroxyphenyl)propionic acid (3HPP). In this work the regulation of the inducible mhp catabolic genes has been studied by genetic and biochemical approaches. The Pr and Pa promoters, which control the expression of the divergently transcribed mhpR regulatory gene and mhp catabolic genes, respectively, show a peculiar arrangement leading to transcripts that are complementary at their 5'-ends. By using Pr-lacZ and Pa-lacZ translational fusions and gel retardation assays, we have shown that the mhpR gene product behaves as a 3HPP-dependent activator of the Pa promoter, being the expression from Pr constitutive and MhpR-independent. DNase I footprinting experiments and mutational analysis mapped an MhpR-protected region, centered at position -58 with respect to the Pa transcription start site, which is indispensable for MhpR binding and in vivo activation of the Pa promoter. Superimposed in the specific MhpR-mediated regulation of the Pa promoter, we have observed a strict catabolite repression control carried out by the cAMP receptor protein (CRP) that allows expression of the mhp catabolic genes when the preferred carbon source (glucose) is not available and 3HPP is present in the medium. Gel retardation assays revealed that the specific activator, MhpR, is essential for the binding of the second activator, CRP, to the Pa promoter. Such peculiar synergistic transcription activation has not yet been observed in other aromatic catabolic pathways, and the MhpR activator becomes the first member of the IclR family of transcriptional regulators that is indispensable for recruiting CRP to the target promoter.

Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10.

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE genes were recruited from another locus. In the 21-kb region upstream from carAa, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 and IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5' portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.

Application of a propionyl coenzyme A synthetase for poly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation in recombinant Escherichia coli.

The genetic operon for propionic acid degradation in Salmonella enterica serovar Typhimurium contains an open reading frame designated prpE which encodes a propionyl coenzyme A (propionyl-CoA) synthetase (A. R. Horswill and J. C. Escalante-Semerena, Microbiology 145:1381-1388, 1999). In this paper we report the cloning of prpE by PCR, its overexpression in Escherichia coli, and the substrate specificity of the enzyme. When propionate was utilized as the substrate for PrpE, a K(m) of 50 microM and a specific activity of 120 micromol. min(-1). mg(-1) were found at the saturating substrate concentration. PrpE also activated acetate, 3-hydroxypropionate (3HP), and butyrate to their corresponding coenzyme A esters but did so much less efficiently than propionate. When prpE was coexpressed with the polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha in recombinant E. coli, a PHA copolymer containing 3HP units accumulated when 3HP was supplied with the growth medium. To compare the utility of acyl-CoA synthetases to that of an acyl-CoA transferase for PHA production, PHA-producing recombinant strains were constructed to coexpress the PHA biosynthetic genes with prpE, with acoE (an acetyl-CoA synthetase gene from R. eutropha [H. Priefert and A. Steinbüchel, J. Bacteriol. 174:6590-6599, 1992]), or with orfZ (an acetyl-CoA:4-hydroxybutyrate-CoA transferase gene from Clostridium propionicum [H. E. Valentin, S. Reiser, and K. J. Gruys, Biotechnol. Bioeng. 67:291-299, 2000]). Of the three enzymes, PrpE and OrfZ enabled similar levels of 3HP incorporation into PHA, whereas AcoE was significantly less effective in this capacity.

Genetic organization and characteristics of the 3-(3-hydroxyphenyl)propionic acid degradation pathway of Comamonas testosteroni TA441.

Comamonas testosteroni TA441 degrades 3-(3-hydroxyphenyl)propionate (3HPP) via the meta pathway. A gene cluster required for degradation of 3HPP was cloned from strain TA441 and sequenced. The genes encoding six catabolic enzymes, a flavin-type hydroxylase (mhpA), extradiol dioxygenase (mhpB), 2-keto-4-pentenoate hydratase (mhpD), acetaldehyde dehydrogenase (acylating) (mhpF), 4-hydroxy-2-ketovalerate aldolase (mhpE) and the meta cleavage compound hydrolase (mhpC), were found in this cluster, encoded in this order. mhpD and mhpF were separated by two genes, orf4 and orf5, which were not necessary for growth on 3HPP. The gene mhpR, encoding a putative transcriptional activator of the IcIR family, was located adjacent to mhpA in the opposite orientation. Disruption of the mhpB or mhpR genes affected growth on 3HPP or trans-3-hydroxycinnamate. The mhpB and mhpC gene products showed high specificity for 3-(2,3-dihydroxyphenyl)propionate (DHPP) and the meta cleavage compound produced from DHPP, respectively.

Removal of volatile acids from synthetic landfill leachate by anaerobic biofilms on drainage aggregates: a laboratory study

Populations of anaerobic bacteria derived from gassing landfills were established on various solid media in laboratory-scale models of landfill drains. Solutions of salts and volatile fatty acids (VFAs), simulating landfill leachates, were circulated through the models for periods of 400 to 800 days and the removal of VFAs and the formation of gas were measured. The ability of the colonized aggregate to remove VFAs from the leachates increased to a maximum over periods of time ranging from 50 to 150 days after the initial colonization phase. The highest rates of VFA removal [3500 mg l–1 (bed vol.) day–1 at a leachate flow rate of 2.59 l d–1 with 16 500 mg l–1 total VFAs] were found at the tops of the columns, but the lower sections were also colonized by bacteria capable of removing VFAs. The initial adaptation of the bacterial population to propionic acid degradation was slower than the adaptation to acetate and butyrate degradation. Increasing the propionate concentration in the leachate from 2500 to 7500 mg l–1 caused a 50% reduction in the rate of removal of VFAs by a bacterial population adapted to a mixture of acetate, propionate and butyrate. The bacterial population eventually adapted to the higher concentrations of propionate. Changing from saturated to unsaturated flow conditions in the drainage models reduced the specific rate of removal of VFAs by 50%.

Removal of volatile acids from synthetic landfill leachate by anaerobic biofilms on drainage aggregates: a laboratory study

Populations of anaerobic bacteria derived from gassing landfills were established on various solid media in laboratory-scale models of landfill drains. Solutions of salts and volatile fatty acids (VFAs), simulating landfill leachates, were circulated through the models for periods of 400 to 800 days and the removal of VFAs and the formation of gas were measured. The ability of the colonized aggregate to remove VFAs from the leachates increased to a maximum over periods of time ranging from 50 to 150 days after the initial colonization phase. The highest rates of VFA removal [3500 mg 1-1 (bed vol.) day -1 at a leachate flow rate of 2.59 1 d-1 with 16 500 mg 1-1 total VFAs] were found at the tops of the columns, but the lower sections were also colonized by bacteria capable of removing VFAs. The initial adaptation of the bacterial population to propionic acid degradation was slower than the adaptation to acetate and butyrate degradation. Increasing the propionate concentration in the leachate from 2500 to 7500 mg 1 -1 caused a 50% reduction in the rate of removal of VFAs by a bacterial population adapted to a mixture of acetate, propionate and butyrate. The bacterial population eventually adapted to the higher concentrations of propionate. Changing from saturated to unsaturated flow conditions in the drainage models reduced the specific rate of removal of VFAs by 50%.

Potentialities of a methanogenic microbial ecosystem adapted to wine distillery wastewaters to degrade volatile fatty acids

The degradation potentiality of Volatile Fatty Acids (VFA), the major metabolic intermediates during methanization of complex organic substrates, has been evaluated. The kinetics of acetic, propionic, butyric and valeric acid degradation by the particular microbial ecosystem are of zero order in relation to the substrate, and acid degradation rate decreass with increasing length of the acid carbon chain. When microorganisms are not active, a propionate or butyrate addition results in a decrease in the acid degradation rate of 40% and 11% respectively, when the initial acid concentrations vary from 20 to 200 m M. On the other hand, if the ecosystem is already degrading propionate or butyrate, an overload of the same VFA (around 50 m M) leads to an increase in the acid degradation rate of 58% and 34%, respectively. Addition of another VFA has a significant inhibiting effect on proiionate or butyrate acetogenic activity a significant inhibiting effect on propionate or butyrate acetogenic activity (e.g. 60 m m of acetic acid decreased propionate and butyrate degradation rates by 68% and 62%,respectively). In all cases, propionate acetogenesis is much more sensitive than butyrate acetogenesis. Propionate degradation is the limiting step of the overall vinasse methanization for the ecosystem studied.