Metabolism of Phosphonoacetate as the Sole Carbon and Phosphorus Source by an Environmental Bacterial Isolate

A gram-negative bacterium isolated from activated sludge was able to utilize up to 25 mM phosphonoacetate as the sole carbon and phosphorus source, with simultaneous excretion of virtually equimolar levels of phosphate. 2-Aminoethylphosphonate was similarly utilized with equivalent growth rates and cellular yields, while 3-aminopropyl-, 4-aminobutyl-, methyl-, ethyl-, and phenylphosphonates served only as phosphorus sources.     

Phosphonate utilization by bacteria.

Bacteria able to use at least one of 13 ionic alkylphosphonates of O-alkyl or O,O-dialkyl alkylphosphonates as phosphorus sources were isolated from sewage and soil. Four of these isolates used 2-aminoethylphosphonic acid (AEP) as a sole carbon, nitrogen, and phosphorus source. None of the other phosphonates served as a carbon source for the organisms. One isolate, identified as Pseudomonas putida, grew with AEP as its sole carbon, nitrogen, and phosphorus source and released nearly all of the organic phosphorus as orthophosphate and 72% of the AEP nitrogen as ammonium. This is the first demonstration of utilization of a phosphonoalkyl moiety as a sole carbon source. Cell-free extracts of P. putida contained an inducible enzyme system that required pyruvate and pyridoxal phosphate to release orthophosphate from AEP; acetaldehyde was tentatively identified as a second product. Phosphite inhibited the enzyme system.     

A role for carbon catabolite repression in the metabolism of phosphonoacetate by Agromyces fucosus Vs2

A strain of Agromyces fucosus, designated Vs2, metabolized a range of organophosphonate compounds as sole phosphorus sources for growth and metabolized phosphonoacetate as a sole carbon, energy and phosphorus source for growth. With phosphonoacetate as the sole phosphorus source and a pyruvate carbon source, transient phosphate release to the medium was observed, in contrast to cultures grown with glucose and phosphonoacetate, where no phosphate release to the medium was observed. Carbon catabolite repression, specifically by means of inducer exclusion of phosphonoacetate, was proposed as the mechanism responsible, and phosphonoacetate hydrolase enzyme assays carried out on cell extracts confirmed that induced phosphonoacetate hydrolase activities were indeed higher in cells grown on pyruvate with phosphonoacetate as sole phosphorus source. This phenomenon has not previously been demonstrated in vivo, and must represent a significant metabolic control of organophosphonate metabolism. The catabolite repression phenomenon was also evident when A. fucosus grew on 2-aminoethylphosphonate as sole phosphorus source, allowing demonstration of a third mode of control for biodegradation of this compound. Excision of stained zymogram gel pieces, followed by tryptic digestion and mass spectrometric analysis, allowed the identification of phosphonoacetate hydrolase-derived peptides.     

牡丹根际溶磷放线菌的筛选及其溶磷特性

通过从牡丹根际土壤中分离筛选溶磷放线菌,得到一株具有较强溶磷能力的菌株PSPSA1,根据形态特征、生理生化特性以及16S rDNA序列分析对菌株进行鉴定,并研究其溶磷遗传稳定性及溶磷特性.菌株PSPSA1被鉴定为白网链霉菌,具有较好的溶磷遗传稳定性.在不同磷源培养液中溶磷量依次为磷酸钙(158.5 mg·L^-1)>磷酸铝(139.9 mg·L^-1)>磷酸铁(127.7 mg·L^-1)>卵磷脂(45.6 mg·L^-1),在无机磷培养液中的溶磷量均与pH呈现显著负相关性,在有机磷培养液中的溶磷量与pH没有显著相关性.在不同碳源条件下的溶磷量依次为乳糖>葡糖糖>麦芽糖>果糖>蔗糖>淀粉>纤维素,在不同氮源条件下的溶磷量依次为蛋白胨>硝酸铵>硫酸铵>硝酸钾>尿素,以葡萄糖为碳源、蛋白胨为氮源时,菌株的溶磷量最高可达202.6 mg·L^-1.土培60 d,单施菌株土壤有效磷含量比对照增加68.2%,菌株与有机肥混施土壤有效磷含量比单施有机肥增加76.7%.表明菌株PSPSA1能够溶解多种难溶磷,在土壤中溶磷效果显著,与有机肥混施其溶磷能力明显提高,有望成为高效生物磷肥的优良菌种.     

The probable metabolic relation between phosphate uptake and energy storages formations under single-stage oxic condition

To investigate the possible biochemical metabolisms for excess phosphate uptake in a sequencing batch reactor (SBR) with single-stage oxic process, which was reported using glucose as the sole carbon source previously, glucose and acetate were fed to two SBRs as the sole carbon source, respectively. The changes of polyhydroxyalkanoates (PHAs), glycogen and the removal of phosphorus were compared between two SBRs. It was observed that the phosphorus removal efficiency was 91.8–94.4% with glucose, and 23.3–28.5% with acetate, although the former showed much lower accumulations/transformations of PHAs. Instead, the former showed a much higher transformation of glycogen. The facts suggested that glycogen could replace PHAs to supply energy for phosphate uptake under the single-stage oxic condition. Furthermore, the possible biochemical metabolisms were proposed to describe the relation between phosphate uptake and energy storages formations under such a single-stage oxic process. Such a process may serve as a prototype for the development of alternative biological and chemical options for phosphate removal from wastewaters.     

Effect of Co‐Substrates on Aerobic Phenol Degradation by Acclimatized and Non‐acclimatized Enrichment Cultures

Aerobic degradation of 7 mmol/L phenol in the presence of alternative carbon sources (7 mmol/L glucose or acetate or 1–2 mmol/L 2‐chlorophenol) was investigated using non‐acclimatized and acclimatized sewage sludges and enrichment cultures. The substrates represented an intermediate of phenol degradation (acetate), an independent substrate (glucose) or a “precursor‐substrate” of phenol degradation (2‐chlorophenol). Bacteria from sewage sludge, not pre‐adapted to phenol (2 mmol/L), rapidly respired acetate and glucose in the presence of phenol, whereas phenol was only bioconverted to any unknown aromatic metabolite after 24 h. In the presence of phenol and 2‐chlorophenol, no removal of both substances was observed when using the unacclimatized sludge. Sludge that was acclimatized to the degradation of phenol showed an initial preference for easily degradable co‐substrates such as glucose or acetate with only a slow concomitant respiration of phenol. Respiration of phenol increased rapidly after the co‐substrates were depleted. The highest phenol degradation rates were 51.6 mmol/L d, when phenol was the sole carbon substrate. Vice versa, phenol was preferentially respired in the presence of a less easily degradable co‐substrate such as 2‐chlorophenol at a rate of around 7 mmol/L d. Further studies with an enrichment culture that was obtained after 7 successive transfers of phenol‐adapted sludge into mineral medium with phenol as the only carbon source indicated that the acetate and glucose‐degrading capabilities were diminished or almost completely lost. In these enrichment cultures, phenol degradation was not affected by the presence of glucose, but glucose was not degraded. In contrary, the presence of acetate slightly slowed down the phenol degradation rate of the enrichment culture. Growth of the microorganisms apparently occurred at the expense of phenol and acetate respiration. The result of this work may be of practical importance in determining the feeding strategy, which is the key factor for most biological wastewater treatment systems. When acetate was present together with phenol in a wastewater, the phenol degradation rates were influenced by acetate, since acetate was an intermediate of phenol degradation. Glucose as an “independent substrate” was apparently degraded by other bacteria via acetate, and in this way it also influenced the phenol degradation rates. Glucose‐degrading bacteria could be “washed out” from the acclimatized sludge during several transfers into mineral medium with phenol as the sole carbon source. If later on, glucose was added again, it remained undegraded and did not influence phenol degradation. 2‐Chlorophenol degradation also requires other bacteria than phenol degraders.     

Effects of different carbon supplements on phosphorus removal in low C/P ratio industrial wastewater

This study focuses on the effects of different carbon supplements on biological phosphorus removal in the optonics and semiconductor industrial wastewater treatment. Experimental results demonstrate that the addition of a carbon source (glucose, acetate, and digester supernatant) improved phosphorus removal effectively. When the COD/P ratios were controlled in the range of 18-20 (using glucose and supernatant as supplement), the acclimated sludge showed more than 98% removal of phosphorus. In addition, different organic carbons induce dissimilar behavior in anaerobic release and aerobic uptake of phosphorus. The glucose supplement induces significant phosphorus release in anaerobic phase and then an increased phosphorus uptake in aerobic phase. The released phosphorus descended in anaerobic phase when acetate and supernatant were added. There was a good linear relationship of first order reaction between initial COD concentration and specific substrate utilization rate in anaerobic phase.     

Nitrogen and Phosphorus in a Stretch of the Guadalupe River,Texas, with Five Main-Stream Impoundments by

Nitrogen and phosphorus were studied in a 168-km stretch of the Guadalupe River that had five main-stream impoundments. Flow through the study area was controlled by releases from these five reservoirs and from Canyon Reservoir, a deep-storage reservoir, located 30 km upstream. Parameters measured monthly on a diel basis at 16 stations were nitrate nitrogen, nitrite nitrogen, ammonia nitrogen, Kjeldahl nitrogen, inorganic phosphate phosphorus, organic phosphate phosphorus, and total phosphate phosphorus.Inorganic nitrogen concentrations observed in this study were as high or higher than that previously reported for other bodies of water. Nitrate nitrogen entered the study area in relatively high concentrations from Comal Springs which was a major source of water for the Guadalupe River. Water from Canyon Reservoir, the other major source of water, was relatively low in nitrate nitrogen. The concentration of nitrate nitrogen was, therefore, dependent in part upon the portion of the total river flow originating from the two sources. Increased discharge from Canyon Reservoir and utilization by plants in areas of high chlorophyll a resulted in low nitrate-nitrogen levels. Retention of water in reservoirs reduced the concentration of nitrate nitrogen due to increased utilization by plants in areas of low flow. Nitrate nitrogen, in general, reached seasonal minima in summer and maxima in winter. Nitrite nitrogen showed considerable variation with no meaningful pattern except that higher concentrations occurred in association with high chlorophyll a and high Kjeldahl nitrogen, regions and periods of low river flow, and large phytoplankton populations. There was no increase in concentration of any form of nitrogen in the vicinity of sewage outfalls and no downstream accrual.Phosphorus levels in the study area were as high or higher than those reported in studies of other bodies of water. Sewage treatment plants at New Braunfels and Seguin, Texas, were major sources of phosphorus to the Guadelupe River. Total phosphate phosphorus was determined to be the most critical phosphate parameter in assessing eutrophication. Seasonally, it ranged from a winter high to a summer low. Concentrations were highest immediately below sewage outfalls and decreased as water progressed downstream. Inorganic-phosphate-phosphorus concentrations showed no clear seasonal trend but were clearly associated with sewage outfalls. Since large standing crops of phytoplankton were observed in areas of low inorganic phosphate phosphorus, it was not considered to limit photosynthesis. Total organic phosphate phosphorus varied seasonally, with high concentrations occurring during the spring and low concentrations in the fall. Total organic phosphate phosphorus showed no correlation with sewage outfalls, but was correlated to a degree with total Kjeldahl nitrogen and chlorophyll a. No consistent pattern of diel fluctuations was evident for any phosphorus or nitrogen compounds analyzed.     

Effect of different carbon sources on the enhanced biological phosphorus removal in a sequencing batch reactor

The effect of the different carbon sources acetate, acetate/glucose or glucose on the enhanced biological phosphorus removal (EBPR) process was studied by experiments under alternating anaerobic–aerobic conditions in one sequencing batch reactor for each carbon source. The glucose was consumed completely within the first 30 min of the anaerobic phase whereas acetate degradation was slow and incomplete. Phosphate was released independently of the carbon source during the whole anaerobic phase. The highest phosphate release (27 mg P l⁻¹) and polyhydroxyalkanoate (PHA) storage (20 mg C g⁻¹ dry matter (DM)) during the anaerobic phase as well as the highest polyphosphate (poly-P) (8 mg P g⁻¹ DM) and glycogen storage (17 mg C g⁻¹ DM) during the aerobic phase were observed with acetate. In contrast to other investigations, glycogen storage did not increase with glucose as substrate but was significantly smaller than with acetate. The PHA composition was also influenced strongly by the carbon source. The polyhydroxyvalerate (PHV) portion of the PHA was maximal 17% for acetate and 82% for glucose. Due to the strong influence of the carbon source on the PHA concentration and composition, PHA storage seems to regulate mainly the phosphate release and uptake. This revised version was published online in July 2006 with corrections to the Cover Date.     

Methanol‐driven enhanced biological phosphorus removal with a syntrophic consortium

The presence of suitable carbon sources for enhanced biological phosphorus removal (EBPR) plays a key role in phosphorus removal from wastewater in urban WWTP. For wastewaters with low volatile fatty acids (VFAs) content, an external carbon addition is necessary. As methanol is the most commonly external carbon source used for denitrification it could be a priori a promising alternative, but previous attempts to use it for EBPR have failed. This study is the first successful report of methanol utilization as external carbon source for EBPR. Since a direct replacement strategy (i.e., supply of methanol as a sole carbon source to a propionic‐fed PAO‐enriched sludge) failed, a novel process was designed and implemented successfully: development of a consortium with anaerobic biomass and polyphosphate accumulating organisms (PAOs). Methanol‐degrading acetogens were (i) selected against other anaerobic methanol degraders from an anaerobic sludge; (ii) subjected to conventional EBPR conditions (anaerobic + aerobic); and (iii) bioaugmented with PAOs. EBPR with methanol as a sole carbon source was sustained in a mid‐term basis with this procedure. Biotechnol. Bioeng. 2013; 110: 391–400. © 2012 Wiley Periodicals, Inc.     

利用污泥提取液制备微生物絮凝剂

以污水厂剩余污泥作为培养基原料,经过一系列处理,探索微生物絮凝剂产生菌的最适发酵培养基配方,结果表明,污泥预处理条件以pH 12碱解条件最优,碳氮源产出量最大,补加8 g/L葡萄糖后灭菌,微生物絮凝剂产生菌LLin6可正常产絮,絮凝率达91.55%。该结果为降低微生物絮凝剂的制备成本,并实现污泥的减量化和污泥资源化利用提供了基础。     

Glyphosate utilization byPseudomonas sp. andAlcaligenes sp. isolated from environmental sources

A total of 21 bacterial cultures were isolated that could utilize glyphosate (N-phosphonomethyl glycine) as a sole source of phosphorus in a mineral salts medium. Sources of inocula for enrichment cultures included aerobic digester liquid, raw sewage, trickling filter effluent, pesticide disposal pit liquid, and soil. Eleven cultures were identified asPseudomonas sp., one asPseudomonas stutzeri, and nine asAlcaligenes sp. Aminomethylphosphonic acid, the major metabolic intermediate of glyphosate degradation in soil, could also serve as a sole phosphorus source for all 21 isolates. Neither glyphosate nor aminomethylphosphonic acid could serve as carbon sources in mineral salts media. Experiments withPseudomonas sp. SG-1 (isolated from aerobic digester liquid) suggested that enzymatic activity responsible for glyphosate degradation was intracellular, inducible, and required the cofactors pyruvate and pyridoxal phosphate. The degradation pathway for glyphosate in this culture may be similar to that previously reported for aminoethylphosphonic acid.     

一株产絮凝剂不动杆菌的筛选及其絮凝特性

从活性污泥中筛选出一株高效的微生物絮凝剂产生菌, 鉴定为鲍曼不动杆菌。蚕豆根尖细胞微核试验未显示该菌株所产絮凝剂具有遗传毒性。该菌产絮凝剂的最佳碳源和氮源分别为葡萄糖和酵母浸出汁, 培养时间为24 h。在絮凝体系中加入Ca2+能明显提高发酵液的絮凝率。在pH为8.0时对高岭土悬浊液和污水具有良好的絮凝效果。     

一株产絮凝剂不动杆菌的筛选及其絮凝特性

从活性污泥中筛选出一株高效的微生物絮凝剂产生菌,鉴定为鲍曼不动杆菌.蚕豆根尖细胞微核试验未显示该菌株所产絮凝剂具有遗传毒性.该菌产絮凝剂的最佳碳源和氮源分别为葡萄糖和酵母浸出汁,培养时间为24 h.在絮凝体系中加入Ca2 能明显提高发酵液的絮凝率.在pH为8.0时对高岭土悬浊液和污水具有良好的絮凝效果.     

Recent developments in the biochemistry and ecology of enhanced biological phosphorus removal

Most of the genes encoding the enzymes involved in polyP synthesis and degradation and in phosphate transport have been studied in various Gram-negative bacteria. Progress has also been made in studying the biochemical mechanisms underlying the process of enhanced biological phosphorus removal (EBPR), in particular in lab-scale systems fed with acetate or acetate plus glucose as the sole carbon and energy sources. By applying 13C-NMR, previous models concerning anaerobic carbon metabolism have been advanced and the role of glycogen in providing reducing equivalents in EBPR is definitely demonstrated. The role of the citric acid cycle in supplying reducing equivalents for the conversion of acetyl-CoA into poly-beta-hydroxybutyrate and poly-beta-hydroxyvalerate has been discussed. An incomplete citric acid cycle has been proposed to provide a small part of the reducing equivalents. Polyphosphate:AMP phosphotransferase and polyphosphatase were readily detectable in EBPR sludge fed with acetate plus glucose, but polyphosphate kinase remained undetected. In a lab-scale EBPR system, fed for several months with only acetate as carbon source, a Rhodocyclus-like bacterium (R6) was highly enriched and is therefore probably responsible for EBPR in systems fed with acetate only. This R6-type bacterium was however also present in other EBPR sludges (but to a lesser extent), and may therefore play an important role in EBPR in general. This organism accumulates polyhydroxyalkanoates anaerobically and polyP under aerobic conditions. Unlike members of the genus Rhodocyclus, bacterium R6 cannot grow phototrophically. Therefore a provisional new genus Candidatus and species Accumulibacter phosphatis was proposed.     

The investigation of effect of organic carbon sources addition in anaerobic–aerobic (low dissolved oxygen) sequencing batch reactor for nutrients removal from wastewaters

The effect of addition of organic carbon sources (acetic acid and waste activated sludge alkaline fermentation liquid) on anaerobic–aerobic (low dissolved oxygen, 0.15–0.45 mg/L) biological municipal wastewater treatment was investigated. The results showed that carbon source addition affected not only the transformations of polyhydroxyalkanoates (PHA), glycogen, nitrogen and phosphorus, but the net removal of nitrogen and phosphorus. The removal efficiencies of TN and TP were, respectively, 61% and 61% without organic carbon source addition, 81% and 95% with acetic acid addition, and 83% and 97% with waste activated sludge alkaline fermentation liquid addition. It seems that the alkaline fermentation liquid of waste biosolids generated in biological wastewater treatment plant can be used to replace acetic acid as an additional carbon source to improve the anaerobic–aerobic (low dissolved oxygen) municipal wastewater nutrients removal although its use was observed to cause a slight increase of effluent BOD and COD concentrations.     

ORGANIC CARBON SUPPLEMENTATION OF STERILIZED MUNICIPAL WASTEWATER IS ESSENTIAL FOR HETEROTROPHIC GROWTH AND REMOVING AMMONIUM BY THE MICROALGA CHLORELLA VULGARIS1

Heterotrophic growth of the microalga Chlorella vulgaris Beij. in synthetic as well as sterilized municipal wastewater of a nonindustrialized city was measured. The city wastewater contained high levels of ammonium and nitrate, medium levels of phosphate, and low levels of nitrite and organic molecules and could not support heterotrophic growth of C. vulgaris. Evaluation of 11 known carbon sources for this microalga that were added to standard synthetic wastewater containing the same levels of nitrogen and phosphorus as the municipal wastewater revealed that the best carbon sources for heterotrophic growth were Na‐acetate and d ‐glucose. These provided the highest growth rates and the largest removal of ammonium. Growth increased with concentration of the supplement to an optimum at 0.12 M Na‐acetate. This carbon source was consumed completely within 10 d of incubation. Higher concentrations inhibited the growth of C. vulgaris. The microalgal populations under heterotrophic growth conditions were one level of magnitude higher than that under autotrophic growth conditions that served as a comparison. No growth occurred in the dark in the absence of a carbon source. Na‐acetate was superior to d ‐glucose. In municipal wastewater, when Na‐acetate or d ‐glucose was added, C. vulgaris significantly enhanced ammonium removal under heterotrophic conditions, and its capacity was equal to ammonium removal under autotrophic growth conditions. This study showed that sterilized wastewater can be treated by C. vulgaris under heterotrophic conditions if supplemented with the appropriate organic carbon source for the microalgae.     

Accumulation of polyhydroxyalkanoates by Microlunatus phosphovorus under various growth conditions

Microlunatus phosphovorus is an activated-sludge bacterium with high levels of phosphorus-accumulating activity and phosphate uptake and release activities. Thus, it is an interesting model organism to study biological phosphorus removal. However, there are no studies demonstrating the polyhydroxyalkanoate (PHA) storage capability of M. phosphovorus, which is surprising for a polyphosphate-accumulating organism. This study investigates in detail the PHA storage behavior of M. phosphovorus under different growth conditions and using different carbon sources. Pure culture studies in batch-growth systems were conducted in shake-flasks and in a bioreactor, using chemically defined growth media with glucose as the sole carbon source. A batch-growth system with anaerobic–aerobic cycles and varying concentrations of glucose or acetate as the sole carbon source, similar to enhanced biological phosphorus removal processes, was also employed. The results of this study demonstrate for the first time that M. phosphovorus produces significant amounts of PHAs under various growth conditions and with different carbon sources. When the PHA productions of all cultivations were compared, poly(3-hydroxybutyrate) (PHB), the major PHA polymer, was produced at about 20–30% of the cellular dry weight. The highest PHB production was observed as 1,421 mg/l in batch-growth systems with anaerobic–aerobic cycles and at 4 g/l initial glucose concentration. In light of these key results regarding the growth physiology and PHA-production capability of M. phosphovorus, it can be concluded that this organism could be a good candidate for microbial PHA production because of its advantages of easy growth, high biomass and PHB yield on substrate and no significant production of fermentative byproducts.     

Fermentation Studies with Streptomyces griseus: I. Carbohydrate Sources for the Production of Protease and Streptomycin

The effects of four carbon sources and inorganic phosphate on the production of streptomycin and protease by a strain of Streptomyces griseus were studied. Protease production was increased in fermentations with comparatively rapid consumption of carbohydrate, and streptomycin was produced under conditions of moderately slow consumption. Starch was consumed more rapidly than glucose, and, in fermentations with starch as a carbon source, good yields of protease were associated with poor yields of streptomycin. The effect of the concentration of inorganic phosphate varied with the sugar source; the rate of consumption of glucose or fructose increased with the addition of inorganic phosphate, and the utilization of starch or maltose was not affected.     

富营养废水中碳氮磷浓度对布朗葡萄藻总脂和总烃的影响

以经过二次过滤的富营养化鱼塘养殖污水为培养液,添加外源的碳、氮、磷元索,研究了污水中不同的外源无机碳、总氮和总磷浓度对布朗葡萄藻(Botryococcus braunii)生物量、总脂和总烃含量的影响.结果表明:(1)以NaHCO3作为碳源,布朗葡萄藻的生物量和总脂含量在外源无机碳浓度为5～10 mg/L时最高,总烃含量在外源无机碳浓度为15mg/L时最高.(2)以KNO3作为氮源,布朗葡萄藻的生物量在总氮浓度为15mg/L时最高,总脂含量在总氮浓度为2mg/L时最高,总烃含量在总氮浓度为20mg/L时最高.(3)以KH2 PO4作为磷源,布朗葡萄藻生物量在总磷浓度为2mg/L时最高,总脂含量和总烃含量在总磷浓度为1.5 mng/L时最高.