Intermediary metabolism of carbon compounds by nitrifying bacteria

Summary The assimilation of¹⁴CO₂ and [2-¹⁴C] acetate, [3-¹⁴C] pyruvate, [5-¹⁴C] -ketoglutarate, [2,3-¹⁴C] succinate, [U-¹⁴C] glutamate and [U-¹⁴C] aspartate was followed in cell suspensions ofNitrosomonas europaea andNitrobacter agilis respectively. There was appreciable incorporation of these substrates even without adding the inorganic nitrogen compounds that are oxidized by these bacteria yielding ATP. In the soluble amino acid fraction most of¹⁴C label was recovered in glutamate while in the protein amino acids a more uniform distribution was found. Acetate was rapidly incorporated to a high level in both nitrifying bacteria while inNitrobacter there was a relatively lower uptake of the other substrates especially succinate. High levels of the NAD malate dehydrogenase and NADP isocitrate dehydrogenase were measured but no significant amounts of the other tricarboxylic acid cycle enzymes or NADH oxidase were found. Glutamate decarboxylase was detected in both organisms and the transferase assay for glutamine synthetase indicated a 30-fold higher activity for this enzyme inNitrobacter. The amino acid composition of the water soluble fraction was determined in both bacteria.     

重组L-门冬酰胺酶工程菌的表达和PEG的化学修饰

目的提高重组L-门冬酰胺酶(rL-ASP)工程菌的表达量,分离纯化rL-ASP并对之进行PEG化学修饰。方法将带有编码rL-ASP的基因的质粒(pKA)导入不同的宿主菌中,挑出高表达菌株,同时优化发酵培养基,分离纯化获得的高纯度rL-ASP再用PEG进行化学修饰,SDS-PAGE检测修饰效果。结果在pH7.0的条件下,宿主菌为JMl09的工程菌pKA/JMl09酶活力最高,三角瓶振摇培养的酶活力可达216×103IU/L;发酵罐发酵培养,酶活力达312×103IU/L。纯化后的rL-ASP比活力为220IU/mg,rL-ASP经过PEG化学修饰生成rL-ASP-PEG,分子量发生改变。结论改变目标蛋白表达的宿主菌和优化发酵工艺,提高了rL-ASP的表达量,纯化的rL-ASP经过PEG化学修饰后分子量增大。     

Metabolism of polyethylene glycol by two anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene glycol. The other isolate, identified as a Bacteroides sp. (strain PG1), metabolized diethylene glycol and polymers of PEG up to an average molecular mass of 20,000 g/mol [PEG 20000; HO-(CH2-CH2-O-)nH]. Both strains produced acetaldehyde as an intermediate, with acetate, ethanol, and hydrogen as end products. In coculture with a Methanobacterium sp., the end products were acetate and methane. Polypropylene glycol [HO-(CH2-CH2-CH2-O-)nH] was not metabolized by either bacterium, and methanogenic enrichments could not be obtained on this substrate. Cell extracts of both bacteria dehydrogenated EG, PEGs up to PEG 400 in size, acetaldehyde, and other mono- and dihydroxylated compounds. Extracts of Bacteroides strain PG1 could not dehydrogenate long polymers of PEG (greater than or equal to 1,000 g/mol), but the bacterium grew with PEG 1000 or PEG 20000 as a substrate and therefore possesses a mechanism for PEG depolymerization not present in cell extracts. In contrast, extracts of D. desulfuricans DG2 dehydrogenated long polymers of PEG, but whole cells did not grow with these polymers as substrates. This indicated that the bacterium could not convert PEG to a product suitable for uptake.     

Lectin-binding assay by polyethylene glycol 8000

A quantitative lectin-binding assay using a precipitation technique and polyethylene glycol 8000 (PEG) as a precipitating agent has been described. Carcinoscorpin, a sialic acid-binding lectin isolated from the hemolymph of Indian horseshoe crab, Carcinoscorpius rotunda cauda, and iodinated fetuin, a sialoglycoprotein, were appropriately incubated as the components of the binding assay. The specific interaction between these two components developed the lectin-glycoprotein-bound complex. This was subsequently precipitated by the addition of PEG together with a coprecipitant gamma-globulin. Radioactivity of the precipitated bound complex was estimated to quantify the binding. The formation of the bound complex was effectively inhibited by a specific sialodisaccharide, O-(N-acetylneuraminyl)-(2----6)-2-acetamido-2-deoxygalactitol, implying the specific interaction for such precipitation. The probable effect of PEG was to stabilize the bound complex, precipitating it along with added gamma-globulin. This was further evident from the prevention of dissociation of the bound complex and increased binding of glycoprotein to the immobilized lectin in the presence of PEG. The assay was also applicable to other sialoglycoproteins such as alpha 1-acid glycoprotein and human chorionic gonadotropin. Moreover, the method yielded a saturation plateau with a characteristic hyperbolic binding curve. The assay was simple, quick, safe, economic, and highly sensitive.     

Immobilization of nitrifying bacteria in porous pellets of urethane gel for removal of ammonium nitrogen from waste-water

Summary The effects of immobilizing materials on the activity of nitrifying bacteria and removal of ammonium nitrogen (NH₄-N) from waste-water by immobilized nitrifying bacteria were investigated using six urethane prepolymers. With a urethane prepolymer containing 2.27% free isocyanate, a high activity yield of nitrifying bacteria was obtained. There was a drastic improvement over the conventional method of immobilization by acrylamide in the activity yield. Inorganic synthetic waste-water was treated at a high loading rate of 0.24 kg N·m^–3·day^–1. The NH₄-N concentration of the effluent could be reduced to 2 mg·1^–1 or less and the removal was 90%. The life of the pellets in terms of activity was at least 120 days. Offprint requests to: T. Sumino     

Ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by microautoradiography-fluorescence in situ hybridization

Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH(4)(+) as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the class Proteobacteria (alpha-Proteobacteria), 23%; gamma-Proteobacteria, 13%; green nonsulfur bacteria (GNSB), 9%; Cytophaga-Flavobacterium-Bacteroides (CFB) division, 2%; and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. alpha- and gamma-Proteobacteria dominated the utilization of [(14)C]acetic acid and (14)C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consuming N-acetyl-D-[1-(14)C]glucosamine (NAG). The GNSB accounted for 9% of the (14)C-amino acid-consuming bacteria and 27% of the [(14)C]NAG-consuming bacteria but did not utilize [(14)C]acetic acid. Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.     

Metabolism of polyethylene glycol by two anaerobic bacteria, Desulfovibrio desulfuricans and a Bacteroides sp.

Two anaerobic bacteria were isolated from polyethylene glycol (PEG)-degrading, methanogenic, enrichment cultures obtained from a municipal sludge digester. One isolate, identified as Desulfovibrio desulfuricans (strain DG2), metabolized oligomers ranging from ethylene glycol (EG) to tetraethylene glycol. The other isolate, identified as a Bacteroides sp. (strain PG1), metabolized diethylene glycol and polymers of PEG up to an average molecular mass of 20,000 g/mol [PEG 20000; HO-(CH2-CH2-O-)nH]. Both strains produced acetaldehyde as an intermediate, with acetate, ethanol, and hydrogen as end products. In coculture with a Methanobacterium sp., the end products were acetate and methane. Polypropylene glycol [HO-(CH2-CH2-CH2-O-)nH] was not metabolized by either bacterium, and methanogenic enrichments could not be obtained on this substrate. Cell extracts of both bacteria dehydrogenated EG, PEGs up to PEG 400 in size, acetaldehyde, and other mono- and dihydroxylated compounds. Extracts of Bacteroides strain PG1 could not dehydrogenate long polymers of PEG (greater than or equal to 1,000 g/mol), but the bacterium grew with PEG 1000 or PEG 20000 as a substrate and therefore possesses a mechanism for PEG depolymerization not present in cell extracts. In contrast, extracts of D. desulfuricans DG2 dehydrogenated long polymers of PEG, but whole cells did not grow with these polymers as substrates. This indicated that the bacterium could not convert PEG to a product suitable for uptake.     

Protein refolding is improved by adding nonionic polyethylene glycol monooleyl ethers with various polyethylene glycol lengths

Protein refolding from bacterial inclusion bodies is a crucial step for the production of recombinant proteins, but the refolding step often results in significantly lower yields due to aggregation. To prevent aggregation, chemical additives are often used. However, the ability of additives to effectively increase refolding yields are protein dependent, and therefore, it is important to understand the manner in which the substructures of additives confer suitable properties on protein refolding. We focused attention on nonionic detergents, the polyethylene glycol monooleyl ether (PGME) series, and systematically studied the influence of two to 90 polyethylene glycol (PEG) lengths of PGMEs on the refolding of pig muscle lactate dehydrogenase (LDH), hen egg white lysozyme, and yeast α‐glucosidase. PGMEs with longer PEG lengths such as PGME20, 50, and 90 suppressed aggregation, and increased refolding yields. Notably, PGME20 increased the LDH yield to 56.7% from 2.5% without additives. According to the refolding kinetic analysis of LDH, compared with PGME50 and 90, the refolding rate constant in PGME20 solutions remained relatively high at a broad range of concentrations because of its weaker steric hindrance of intramolecular interactions involved in folding, leading to a preference for refolding over aggregation. These findings should provide basic guidelines to identify appropriate PEG‐based nonionic detergents for protein refolding.     

Alterations in phospholipid polymorphism by polyethylene glycol

Summary The fusogen polyethylene glycol is shown to alter the polymorphism of dimyristoyl phosphatidylcholine, soybean phosphatidylethanolamine, bovine phosphatidylserine, egg phosphatidylcholine/cholesterol mixture, dilinoleoylphosphatidylethanolamine/palmitoyl-oleoylphosphatidylcholine mixture, and egg lysolecithin. Suspension of these lipids in 50% polyethylene glycol (mol wt=6000) reduces both the lamellar and the hexagonal II repeat spacings as measured by X-ray diffraction. An increase in the gel to liquid crystalline and bilayer to hexagonal transition temperatures are observed by freeze-fracture, X-ray diffraction, differential scanning calorimetry and³¹P NMR. Freeze-fracture electron micrographs revealed different bilayer defects depending on the physical states of the lipid. Lipidic particles in mixtures containing unsaturated phosphatidylethanolamine is eliminated. Some of the influences of polyethylene glycol on lipids may be explained by its dehydrating effect. However, other nonfusogenic dehydrating agents failed to produce similar results. These findings are consistent with the proposal that close bilayer contact and the formation of bilayer defects are associated with the fusogenic properties of polyethylene glycol.     

Phosphate requirements of the nitrifying bacteria

The influence of the phosphate concentration on the specific growth rate and the duration of lag has been studied inNitrobacter winogradskyi andNitrosomonas europaea.The optimum phosphate concentration range for the specific growth rate was 10 to 30mm forNitrobacter and 10 to 100mm forNitrosomonas. In this range the lag was least. Depletion of the cell-P does not affect the relation between specific growth rate and phosphate concentration while the lag seems to increase as cell-P depletion proceeds.     

Ammonium removal performance of anaerobic ammonium-oxidizing bacteria immobilized in polyethylene glycol gel carrier

Anaerobic ammonium-oxidizing (anammox) bacteria were immobilized in polyethylene glycol gel carriers. A small amount of seed sludge [0.24% (w/v)] was entrapped in the carriers, and continuous feeding tests were performed. Nitrogen removal activity increased gradually, reaching 3.7 kg N/m(3) reactor per day on day 67. The average of nitrogen conversion rate was calculated as 3.4 kg N/m(3) reactor per day. Microscopic examination clearly showed that small red clusters formed in the gel carrier. Moreover, fluorescence in situ hybridization analysis revealed that these clusters consisted of anammox bacteria. From real-time polymerase chain reaction analysis, the growth of anammox bacteria in the gel carriers was clearly shown by increased concentration of 16S rRNA gene of planctomycete from 4.3 x 10(8) to 4.2 x 10(9) copies/ml between days 41 and 55. To determine the effects of inoculation on the start-up of the reactor, the amount of seed sludge in the gel carrier was varied and it was found that the start-up period could be reduced to as little as 25 days when a sludge concentration of 1.4% (w/v) was used. This is the first report of successful immobilization and cultivation of anammox bacteria in a gel carrier.     

Improved stability of aerobic granules by selecting slow-growing nitrifying bacteria

This study investigated the feasibility of improving the stability of aerobic granules through selecting slow-growing nitrifying bacteria. For this purpose, four sequencing batch reactors were operated at different substrate N/COD ratios ranging from 5/100 to 30/100. Results showed that aerobic granules formed in all four reactors, and aerobic granulation was a gradual process evolving from the dispersed seed sludge to mature and stable granules, and the whole granulation process could be divided into three phases, i.e. acclimation phase, granulation followed by granule maturation. The observed growth rate and mean size of mature aerobic granules were found to decrease as the substrate N/COD ratio was increased, while nitrifying population was enriched markedly in aerobic granules developed at high substrate N/COD ratios. The enriched nitrifying population in aerobic granules was responsible for the observed low growth rate of aerobic granules. It seems certain that the substrate N/COD ratio is an important factor in selecting nitrifying bacteria in aerobic granules. Aerobic granules with low growth rates showed strong structure and good settleability in terms of specific gravity, SVI and cell hydrophobicity that further lead to high stability as compared to those having high growth rates. This study demonstrated that the selection of slow-growing nitrifying bacteria through controlling substrate N/COD ratio would be a useful strategy for improving the stability of aerobic granules.