Anaerobic degradation of veratrylglycerol-beta-guaiacyl ether and guaiacoxyacetic acid by mixed rumen bacteria.

Veratrylglycerol-beta-guaiacyl ether (0.2 g/liter), a lignin model compound, was found to be degraded by mixed rumen bacteria in a yeast extract medium under strictly anaerobic conditions to the extent of 19% within 24 h. Guaiacoxyacetic acid, 2-(o-methoxyphenoxy)ethanol, vanillic acid, and vanillin were detected as degradation products of veratrylglycerol-beta-guaiacyl ether by thin-layer chromatography, gas chromatography, and gas chromatography-mass spectrometry. Guaiacoxyacetic acid (0.25 g/liter), when added into the medium as a substrate, was entirely degraded within 36 h, resulting in the formation of phenoxyacetic acid, guaiacol, and phenol. These results suggest that the beta-arylether bond, an important intermonomer linkage in lignin, can be cleaved completely by these rumen anaerobes.     

Degradation of benzyl ether bonds of lignin by ruminal microbes

We examined microbial activity in the rumen to cleave benzyl ether bonds of lignin model compounds that fluoresced when the bonds were cleaved. 4-Methylumbelliferone veratryl ether dimer was degraded completely within 8 h even in the presence of fungicidal antibiotics, but no significant degradation occurred with bactericidal antibiotics. Degradation of a phenolic beta-O-4 trimer incorporating 4-methylumbelliferone by a benzyl ether linkage was stimulated by ruminal microbes, although its corresponding non-phenolic model compound, 1-(4-ethoxy-3-methoxyphenyl)-1-O-(4-methylumbelliferyl)-2-(2-methoxyp henoxy)-3-propanol, was not degraded. A coniferyl dehydrogenation polymer bearing fluorescent beta-O-4 benzyl ether that contains both phenolic and non-phenolic benzyl ether bonds was partially degraded (about 20%) in 48 h. These results suggest that ruminal microbes decompose benzyl ether linkages of lignin polymers under anaerobic conditions.     

Degradation of Dehydrodivanillin by Anaerobic Bacteria from Cow Rumen Fluid

Dehydrodivanillin (DDV; 0.15 g/liter) was biodegradable at 37°C under strictly anaerobic conditions by microflora from cow rumen fluid to the extent of 25% within 2 days in a yeast extract medium. The anaerobes were acclimated on DDV for 2 weeks, leading to DDV-degrading microflora with rates of degradation eight times higher than those initially. Dehydrodivanillic acid and vanillic acid were detected in an ethylacetate extract of a DDV-enriched culture broth by thin-layer, gas, and high-performance liquid chromatographies and by mass spectrometry.     

Effect of harvest date and nitrogen fertilization rate on the nutritive value of amaranth forage (Amaranthus hypochondriacus)

This study was conducted to assess effects of harvest date (i.e., 40 and 60 d after planting) and N fertilization rate (i.e., 120, 180, 240 kg N/ha) on the nutritive value of amaranth forage (Amaranthus hypochondriacus) using a factorial experiment with a randomized complete block design. The content of dry matter (DM), crude protein (CP), true protein (TP), ether extract (EE), water soluble carbohydrates (WSC), ash-free neutral detergent fiber (NDFom), ash-free acid detergent fiber (ADFom), lignin(sa), ash, Ca, P, Na, K, oxalic acid and nitrate were determined. Soluble CP (SP) and protein fractions non-protein N (A), true protein rapidly degraded in the rumen (B₁), true protein degraded in the rumen at a moderate rate (B₂), true protein associated with the cell wall and slowly degraded in the rumen (B₃) and acid detergent insoluble CP (C) were measured according to the Cornell Net Carbohydrate and Protein System. In vitro gas production (IVGP), OM disappearance (OMD) and NDFom disappearance (NDFD) were determined using a gas production technique. Results showed that the later harvest date increased (P<0.05) DM, EE, WSC, NDFom, ADFom, lignin(sa), B₃ and C; while CP, TP, ash, Ca, P, K, SP, A, B₁, B₂, nitrate, total and soluble oxalic acid, IVGP, b (i.e., gas production from the insoluble fermentable fractions at 120 h), c (i.e., rate of gas production during incubation), OMD and NDFD decreased (P<0.05). With increasing N fertilization rate, CP, TP, EE, P, nitrate, oxalic acid, SP, A, b, OMD and NDFD increased (P<0.05), however B₂ declined (P<0.05). Increasing N fertilization increased yield, CP concentration and nutrient digestibility. At 40 d after planting use of amaranth forage as a ruminant feed is limited due to its high nitrate content. However, at 60 d, although a depression in digestibility and CP content occurred, this forage has the potential as a ruminant feed due to the much lower nitrate levels.     

Effects of lignin on the anaerobic degradation of (ligno) cellulosic wastes by rumen microorganisms

Summary There appeared to be a clear correlation between the lignin content (% of TS) of several waste and natural materials and their degradability by rumen microorganisms. Materials with lignin contents higher than 25% were not degraded within 72 h. The effects of Kraft pine lignin and some lignin monomers on filter paper degradation, methane production and CMCase activity were tested. Testing these compounds in concentrations comparable to natural conditions showed minor effects. At higher concentrations p-coumaric acid strongly inhibited cellulose degradation and methane production in batch cultures. Influence of lignin compounds on degradation is discussed in relation to structural effects and enzyme or growth inhibition.     

Biodegradation of Green HE4B: Co-substrate effect,biotransformation enzymes and metabolite toxicity analysis

A high exhaust reactive dye, Green HE4B (GHE4B) was 98% degraded in nutrient medium by Pseudomonas desmolyticum NCIM 2112 (pd2112) within 72 h at static condition. Decolorization time in synthetic 10 g/l molasses. Addition of 5 g/l peptone to NaCl medium had reduced decolorization time from 108 to 72 h. Beef extract do not contribute more to the inducing effect of peptone, however it is a good co-substrate in sucrose or urea containing NaCl medium. Intracellular lignin peroxidase (Lip), laccase and tyrosinase activities were induced by 150, 355 and 212%, respectively till maximum dye removal took place. Aminopyrine N-demethylase (AND) and dichlorophenol indophenol reductase (DCIP-reductase) activities in pd2112 were induced by 130 and 20%, respectively at 72 h of incubation during GHE4B decolorization. By high performance liquid chromatography (HPLC) analysis, 4-hydroxybenzene sulfonic acid and 4-amino, 6-hydroxynaphthalene 2-sulfonic acids were identified as metabolites formed during 24–72 h incubation. Fourier transform infrared spectroscopy (FTIR) analysis supports the formation of these aromatic amines. pd2112, aerobically degraded GHE4B metabolites (formed at static condition) showing stationary phase of 6 days. There was no germination inhibition of Sorghum bicolor and Triticum aestivum by GHE4B metabolites at 3,000 ppm concentration however untreated dye showed germination inhibition at the same concentration. GHE4B metabolites did not show any microbial toxicity at 10,000 ppm concentration.     

Studies on the possibility of histidine biosynthesis from histidinol, imidazolepyruvic acid, imidazoleacetic acid, and imidazolelactic acid by mixed ruminal bacteria, protozoa, and their mixture in vitro

The possibility of histidine (His) synthesis using a main biosynthetic pathway involving histidinol (HDL) and also the recycling capability of imidazolic compounds such as imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) to produce His were investigated using mixed ruminal bacteria (B), protozoa (P), and a mixture of both (BP) in an in vitro system. Rumen microorganisms were anaerobically incubated at 39 degrees C for 18 h with or without each substrate (2 mM) mentioned. His and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by high-performance liquid chromatography. B, P, and BP suspensions failed to show His synthesizing ability when incubated with HDL. His was synthesized from ImPA by B, P, and BP. Expressed in units "per gram of microbial nitrogen (MN)", ImPA disappearance was greatest in B (72.7 micromol/g MN per hour), followed by BP (33.13 micromol/g MN per hour) and then P (18.6 micromol/g MN per hour) for the 18-h incubation period. The production of His from ImPA in B (240.0, 275.9, and 261.2 micromol/g MN in 6, 12, and 18 h incubation, respectively) was about 3.5 times higher than that in P (67.3, 83.8, and 72.7 micromol/g MN in 6, 12, and 18 h incubation, respectively). Other metabolites produced from ImPA were ImLA, ImAA, histamine (HTM), and urocanic acid (URA), found in all microbial suspensions. ImLA as a substrate remained without diminution in all microbial suspensions. Although ImAA was found to be degraded to a small extent (3.4-6.3%) only after 18 h incubation, neither His nor other metabolites were detected on the chromatograms. These results have been demonstrated for the first time in rumen microorganisms and suggest that His may be an essential amino acid for rumen microorganisms.     

Toxicity of organic extraction reagents to anaerobic bacteria

Various forms of liquid-liquid extraction systems are being developed to separate products, such as ethanol and volatile fatty acids (VFA), from fermentation liquids, since distillation is energetically expensive. Continuous extraction is advantageous, as product inhibition of the fermentation is minimized. However, some extraction solvents may be toxic or inhibitory to microorganisms.Thirty organic chemicals were examined by means of a small scale (60 mL) batch fermentation bioassay procedure for their toxicity to a commercial inoculum (Methanobac, W.B.E. Ltd.), which was a mixed culture of facultatively anaerobic, acid-producing bacteria. Gas production, pH change of medium, and the concentrations of ethanol, VFA, and lactic acid were measured after 75 h growth. The optimum experimental conditions for toxicity testing were alfalfa as substrate (2 g), a buffered nutrient medium (pH 6.8), "Methanobac" inoculum (10 mL), and test chemicals at levels between 10 and 100 muL/mL.Thirteen chemicals were nontoxic, and included the paraffins (C(6)-C(12)), phthalates, organophosphorus compounds, Freon 113 (1,1,2-trichloro-1,2,2-trifluoro ethane), Aliquat 336 (tricaprylylmethyl ammonium chloride), di-isoamyl ether, and trioctylamine. Other amine extractants were partially toxic. Alcohols (C(5)-C(12)), ketones (C(5)-C(8)), benzene derivatives, isoamyl acetate, and di-isopropyl ether were toxic. Generally, the chemicals were not toxic unless present at levels in excess of that expected to be required to saturate the aqueous phase.Total gas production was a good indicator of toxicity even within 24 h, but the presence of homofermentative (nongas producing) lactic acid bacteria complicated interpretation."Methanobac" inoculum was compared with an inoculum derived from a rumen culture for four test chemicals. The results were essentially the same. However, the toxicity of a chemical to bacteria is likely to vary considerably between bacterial species.     

Displacement of Escherichia coli O157:H7 from rumen medium containing prebiotic sugars

A fed-batch, anaerobic culture system was developed to assess the behavior of Escherichia coli O157:H7 in a rumen-like environment. Fermentation medium consisted of either 50% (vol/vol) raw or sterile rumen fluid and 50% phosphate buffer. Additional rumen fluid was added twice per day, and samples were removed three times per day to simulate the exiting of digesta and microbes from the rumen environment under typical feeding regimens. With both types of medium, anaerobic and enteric bacteria reached 10(10) and 10(4) cells/ml, respectively, and were maintained at these levels for at least 5 days. When a rifampin-resistant strain of E. coli O157:H7 was inoculated into medium containing raw rumen fluid, growth did not occur. In contrast, when this strain was added to sterile rumen fluid medium, cell densities increased from 10(6) to 10(9) CFU/ml within 24 h. Most strains of E. coli O157:H7 are unable to ferment sorbitol; therefore, we assessed whether the addition of sorbitol as the only added carbohydrate could be used to competitively exclude E. coli O157:H7 from the culture system. When inoculated into raw rumen broth containing 3 g of sorbitol per liter, E. coli O157:H7 was displaced within 72 h. The addition of other competitive sugars, such as L-arabinose, trehalose, and rhamnose, to rumen medium gave similar results. However, whenever E. coli O157:H7 was grown in sterile rumen broth containing sorbitol, sorbitol-positive mutants appeared. These results suggest that a robust population of commensal ruminal microflora is required to invoke competitive exclusion of E. coli O157:H7 by the addition of "nonfermentable" sugars and that this approach may be effective as a preharvest strategy for reducing carriage of E. coli O157:H7 in the rumen.     

Degradation of estrogens by Rhodococcus zopfii and Rhodococcus equi isolates from activated sludge in wastewater treatment plants

We have isolated four strains of Rhodococcus which specifically degrade estrogens by using enrichment culture of activated sludge from wastewater treatment plants. Strain Y 50158, identified as Rhodococcus zopfii, completely and rapidly degraded 100 mg of 17beta-estradiol, estrone, estriol, and ethinyl estradiol/liter, as demonstrated by thin-layer chromatography and gas chromatography-mass spectrometry analyses. Strains Y 50155, Y 50156, and Y 50157, identified as Rhodococcus equi, showed degradation activities comparable with that of Y 50158. Using the random amplified polymorphism DNA fingerprinting test, these three strains were confirmed to have been derived from different sources. R. zopfii Y 50158, which showed the highest activity among these four strains, revealed that the strain selectively degraded 17beta-estradiol during jar fermentation, even when glucose was used as a readily utilizable carbon source in the culture medium. Measurement of estrogenic activities with human breast cancer-derived MVLN cells showed that these four strains each degraded 100 mg of 17beta-estradiol/liter to 1/100 of the specific activity level after 24 h. It is thus suggested that these strains degrade 17beta-estradiol into substances without estrogenic activity.     

Isolation and characterization of an anaerobic dehydrodivanillin-degrading bacterium

A novel, strictly anaerobic, gram-negative, non-spore-forming, fusiform, rod-shaped bacterium having high dehydrodivanillin (DDV)-degrading activity was isolated from cow ruminal fluid. This strain degraded a range of six main lignin-related compounds such as DDV, ferulic acid, dehydrodiisoeugenol, guaiacoxyacetic acid, vanillin, and veratrylglycerol-beta-guaiacyl ether to the extent of 14 to 83% within 2 days under strictly anaerobic conditions. As DDV degradation intermediates, three aromatic compounds (dehydrodivanillic acid, vanillic acid, and 5-carboxyvanillic acid) and two alicyclic compounds (cyclohexanecarboxylic acid and cyclohexanol) were detected by thin-layer, high-performance liquid, and gas chromatography and mass spectrometry. The addition of 1% glucose and peptone in a synthetic medium stimulated growth of the strain but slowed down DDV degradation. The presence of 0.1% yeast extract increased both cell growth and DDV degradation. The growth yield in defined medium was 151.5 g (dry weight) of cells per mol of DDV utilized. Characterization of the strain indicated that it was distinct from known Fusobacterium and Clostridium species. The bacterium was easily induced to form protoplasts after treatment with either penicillin or lysozyme. The frequencies of protoplast formation and regeneration in the strain were 94 and 18%, respectively.     

Isolation and characterization of an anaerobic dehydrodivanillin-degrading bacterium.

A novel, strictly anaerobic, gram-negative, non-spore-forming, fusiform, rod-shaped bacterium having high dehydrodivanillin (DDV)-degrading activity was isolated from cow ruminal fluid. This strain degraded a range of six main lignin-related compounds such as DDV, ferulic acid, dehydrodiisoeugenol, guaiacoxyacetic acid, vanillin, and veratrylglycerol-beta-guaiacyl ether to the extent of 14 to 83% within 2 days under strictly anaerobic conditions. As DDV degradation intermediates, three aromatic compounds (dehydrodivanillic acid, vanillic acid, and 5-carboxyvanillic acid) and two alicyclic compounds (cyclohexanecarboxylic acid and cyclohexanol) were detected by thin-layer, high-performance liquid, and gas chromatography and mass spectrometry. The addition of 1% glucose and peptone in a synthetic medium stimulated growth of the strain but slowed down DDV degradation. The presence of 0.1% yeast extract increased both cell growth and DDV degradation. The growth yield in defined medium was 151.5 g (dry weight) of cells per mol of DDV utilized. Characterization of the strain indicated that it was distinct from known Fusobacterium and Clostridium species. The bacterium was easily induced to form protoplasts after treatment with either penicillin or lysozyme. The frequencies of protoplast formation and regeneration in the strain were 94 and 18%, respectively.     

Involvement of extracellular dilignols in lignification during tracheary element differentiation of isolated Zinnia mesophyll cells

During differentiation of isolated Zinnia mesophyll cells into tracheary elements (TEs), lignification on TEs progresses by supply of monolignols not only from TEs themselves but also from surrounding xylem parenchyma-like cells through the culture medium. However, how lignin polymerizes from the secreted monolignols has not been resolved. In this study, we analyzed phenol compounds in culture medium with reversed-phase HPLC, gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry, and found 12 phenolic compounds including coniferyl alcohol and four dilignols, i.e. erythro-guaiacylglycerol-beta-coniferyl ether, threo-guaiacylglycerol-beta-coniferyl ether, dehydrodiconiferyl alcohol and pinoresinol, in the medium in which TEs were developing. Coniferyl alcohol applied to TE-inductive cultures during TE formation rapidly disappeared from the medium, and caused a sudden increase in dilignols. Addition of the dilignols promoted lignification of TEs in which monolignol biosynthesis was blocked by an inhibitor of phenylalanine anmmonia-lyase (PAL), L-alpha-aminooxy-beta-phenylpropionic acid (AOPP). These results suggested that dilignols can act as intermediates of lignin polymerization.     

Degradation of labelled lignins and veratrylglycerol-beta-guaiacyl ether by Acinetobacter sp

Acinetobacter sp. evolved 14CO2 from 14C-(ring)DHP lignin and 14C-teakwood lignin. Veratrylglycerol-beta-guaiacyl ether, a lignin model compound with beta-o-4 linkage was cleaved by Acinetobacter sp. Veratrylglycerol-beta-guaiacyl ether into 2(o-methoxyphenoxy) ethanol and veratrylalcohol 2(o-methoxyphenoxy) ethanol was degraded to guaiacol and then to catechol whereas veratrylalcohol was converted to veratraldehyde, veratric acid, vanillic acid, protocatechuic acid and catechol. Both catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase were detected in veratrylglycerol-beta-guaiacyl ether grown cultures.     

Bonding of hydroxycinnamic acids to lignin: ferulic and p-coumaric acids are predominantly linked at the benzyl position of lignin, not the beta-position, in grass cell walls.

A suspension in dichloromethane-water (18:1, v/v) of various fractions containing hydroxycinnamic acid ester-ether bridges between lignin and polysaccharides prepared from cell walls of matured oat (Avena sativa L.) intemodes, and a solution of their acetates in the same solvent, were treated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). This reagent selectively cleaves benzyl ether and ester linkages of negatively charged aromatic nuclei. The sample treated with DDQ was directly hydrolysed either under mild (1 M NaOH, overnight at 37 degrees C) or severe (4 M NaOH, for 2 h at 170 degrees C) conditions. The hydroxycinnamic acids released in the hydrolysate were methylated with diazomethane and analysed quantitatively using gas chromatography. Significant portions of ether linkages between hydroxycinnamic acids and lignin were cleaved with DDQ, which suggests that most of the hydroxycinnamic acids were ether-linked at the benzyl position, and not the beta-position, of the lignin side chain as previously claimed.     

Production of tyrosine and other aromatic compounds from phenylalanine by rumen microorganisms

Summary Rumen contents from three fistulated Japanese native goats fed Lucerne hay cubes (Medicago sativa) and concentrate mixture were collected to prepare the suspensions of mixed rumen bacteria (B), mixed protozoa (P) and a combination of the two (BP). Microbial suspensions were anaerobically incubated at 39°C for 12h with or without 1 MM ofl-phenylalanine (Phe). Phe, tyrosine (Tyr) and other related compounds in both supernatant and microbial hydrolysates of the incubations were analyzed by HPLC. Tyr can be produced from Phe not only by rumen bacteria but also by rumen protozoa. The production of Tyr during 12h incubation in B (183.6 mol/g MN) was 4.3 times higher than that in P. One of the intermediate products between Phe and Tyr seems to bep-hydroxyphenylacetic acid. The rate of the net degradation of Phe incubation in B (76.O mol/g MN/h) was 2.4 times higher than in P. In the case of all rumen microorganisms, degraded Phe was mainly (>53%) converted into phenylacetic acid. The production of benzoic acid was higher in P than in B suspensions. Small amount of phenylpyruvic acid was produced from Phe by both rumen bacteria and protozoa, but phenylpropionic acid and phenyllactic acid were produced only by rumen bacteria.     

In sacco rumen disappearance of condensed tannins, fiber, and nitrogen from herbaceous native Texas legumes in goats

Condensed tannins (CT) can play a role in rumen protein and fiber degradability, especially in legumes high in CT. In order to better understand their potential role in ruminant nutrition, three legume species native to Texas, Acacia angustissima var. hirta (prairie acacia) (288.0 g/kg neutral detergent fiber (NDFom), 40.9 g/kg N), Desmodium paniculatum (panicled tick-clover) (479.7 g/kg NDFom, 24.8 g/kg N), and Lespedeza procumbens (trailing bush-clover) (401.0 g/kg NDFom, 21.7 g/kg N) were studied to determine in sacco disappearance rates of key nutritional components compared to that of Medicago sativa (alfalfa) (226.8 g/kg NDFom, 34.6 g/kg N). Herbage was incubated in rumen-cannulated goats fed a basal diet of Sorghum bicolor×S. sudanense (sorghum-Sudan) hay, with disappearance measured at 0, 4, 8, 16, 28, 48 and 96 h. Among the native legumes, the highest CT concentrations were measured in prairie acacia (263 g CT/kg DM foliage) and the lowest (120 g CT/kg DM) in trailing bush-clover. The lowest concentrations of acid detergent fiber (ADFom), NDFom, and sulfuric acid lignin (lignin(sa)) were measured in prairie acacia, the first two fractions being comparable to alfalfa. Proportion remaining was calculated for CT, ADFom, lignin(sa), NDFom, and N for 0, 24 and 48 h of rumen incubation. Disappearance parameters were measured for ADFom, lignin(sa), NDFom and N for the three native legumes and compared to alfalfa. Alfalfa had the highest disappearance of all degradable fractions except lignin(sa). Potential disappearance (PD) fraction for ADFom, lignin(sa) and N were lower for the native legumes versus alfalfa. No differences in N proportion remaining at 24 and 48 h occurred in the native legumes despite differences in protein-bound CT proportion remaining at those same times. Of the native legumes studied, prairie acacia shows the greatest potential for contributing rumen-escape protein, suggesting it may be a candidate for further development as a pasture and rangeland renovation legume.     

In Vitro Catabolism of Histidine by Mixed Rumen Bacteriaand Protozoa

An in vitro study was conducted to examine the metabolism of histidine (His) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 2 mm each of His, or histamine (HTM), or 1 mm urocanic acid (URA) at 39°C for 12 h. His and other related compounds in both supernatant and microbial hydrolysates were analyzed by HPLC. After 6- and 12-h incubations, the net degradation of His was 26.1% and 51.7% in B, 13.5% and 20.9% in P, and 21.7% and 46.0% in BP, respectively. The rate of the net degradation of His in B (98.0 μmol/g microbial nitrogen/h) was about 2.6 times higher than that of P during a 12-h incubation period. His was found to be degraded into urocanic acid (URA), imidazolelactic acid (ImLA), imidazoleacetic acid (ImAA), and histamine (HTM). Of these degraded His was mainly converted into URA in all microbial suspensions. The production of ImLA and ImAA was higher in B than in P suspensions, whereas the production of HTM was higher in P than in B suspensions. From these results, the existence of diverse catabolic routes of His in rumen microorganisms was indicated. Received: 23 May 2000 / Accepted: 31 July 2000     

Biodegradation of ether pollutants by Pseudonocardia sp. strain ENV478

A bacterium designated Pseudonocardia sp. strain ENV478 was isolated by enrichment culturing on tetrahydrofuran (THF) and was screened to determine its ability to degrade a range of ether pollutants. After growth on THF, strain ENV478 degraded THF (63 mg/h/g total suspended solids [TSS]), 1,4-dioxane (21 mg/h/g TSS), 1,3-dioxolane (19 mg/h/g TSS), bis-2-chloroethylether (BCEE) (12 mg/h/g TSS), and methyl tert-butyl ether (MTBE) (9.1 mg/h/g TSS). Although the highest rates of 1,4-dioxane degradation occurred after growth on THF, strain ENV478 also degraded 1,4-dioxane after growth on sucrose, lactate, yeast extract, 2-propanol, and propane, indicating that there was some level of constitutive degradative activity. The BCEE degradation rates were about threefold higher after growth on propane (32 mg/h/g TSS) than after growth on THF, and MTBE degradation resulted in accumulation of tert-butyl alcohol. Degradation of 1,4-dioxane resulted in accumulation of 2-hydroxyethoxyacetic acid (2HEAA). Despite its inability to grow on 1,4-dioxane, strain ENV478 degraded this compound for > 80 days in aquifer microcosms. Our results suggest that the inability of strain ENV478 and possibly other THF-degrading bacteria to grow on 1,4-dioxane is related to their inability to efficiently metabolize the 1,4-dioxane degradation product 2HEAA but that strain ENV478 may nonetheless be useful as a biocatalyst for remediating 1,4-dioxane-contaminated aquifers.     

Genome-based metabolic engineering of Mannheimia succiniciproducens for succinic acid production

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO2-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.