Synthesis of intracellular storage polymers by Amaricoccus kaplicensis, a tetrad forming bacterium present in activated sludge

AIMS: The study investigated the physiology of Amaricoccus kaplicensis to determine whether it could outcompete polyphosphate accumulating bacteria in activated sludge systems removing phosphorus, by preferentially assimilating substrates in the anaerobic stages of these processes. METHODS AND RESULTS: The storage processes were investigated under anaerobic, anoxic and aerobic conditions in both batch and periodically fed cultures in an aerobic sequencing batch reactor (SBR). Amaricoccus kaplicensis showed a high capacity for storing aerobically large amounts of acetate as poly beta-hydroxybutyrate (PHB) at high rates. However, no acetate assimilation under anaerobic conditions and very slow assimilation under anoxic conditions could be detected. CONCLUSION: Amaricoccus kaplicensis in pure culture does not behave as polyphosphate accumulating bacteria competitor; therefore it is difficult to understand why anaerobic/aerobic systems often contain such large numbers of Amaricoccus cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Amaricoccus kaplicensis is probably not responsible for the failure of activated sludge systems removing phosphorus, and other organisms capable of anaerobic substrate assimilation should be sought.     

Metabolic transformations and characterisation of the sludge community in an enhanced biological phosphorus removal system

Enhanced biological phosphorus removal was performed in a continuous laboratory-scale two-reactor system with sludge recirculation over a 75-day period. Influent wastewater was a synthetic medium based on acetate, and the sludge age was kept at 12 days. The adapted sludge stored poly-β-hydroxyalkanoic acids (PHA) in the anaerobic reactor with a conversion ratio of 1.45 PHA/acetic acid (based on chemical O₂ demand: COD/COD) and gave ratio of a phosphate-P release to acetic acid uptake of 0.51 P/CH₃COOH (w/w). Fractionation of anaerobic and aerobic sludges showed that the main part of phosphorus taken up, was eluted in the trichloroacetic acid fraction indicating that it was polyphosphate. A total of 60% of the phosphorus in the aerobic sludge was solubilized in the trichloroacetic acid fraction, whereas this fraction accounted for only 32% of the phosphorus in the anaerobic sludge. Only 4% of the total phosphorus in the aerobic sludge and 2% in the anaerobic sludge was found in the EDTA fraction, indicating low amounts of metal-bound phosphates. Isolation on acetate-based agar medium showed that Acinetobacter strains were present in the sludge. However, a more complete analysis of the bacterial community of the sludge was obtained by creating a clone library based on the 16S rRNA gene. A total of 51 partial clone sequences were phylogenetically evaluated. The predominating group was found in the high-(G+C) (mol%) gram-positive bacterial subphylum (31% of the sequenced clones), while the gamma proteobacteria only constituted 9.8% of the clones. Received: 12 June 1997 / Received revision: 26 September 1997 / Accepted: 28 September 1997     

Production of polyhydroxybutyrate by activated sludge performing enhanced biological phosphorus removal

In this study, polyhydroxybutyrate (PHB) – a biodegradable plastics material – was produced by activated sludge performing enhanced biological phosphorus removal (EBPR) in batch experiments under anaerobic, aerobic and anaerobic/aerobic conditions. Under anaerobic conditions, the maximum PHB content of the dry biomass was 28.8% by weight, while under aerobic or anaerobic/aerobic conditions, the maximum PHB content was about 50%. The PHB production rate with respect to the volatile suspended solids (VSS) was: (i) 70 mg/(g VSS) h under aerobic conditions that followed anaerobic conditions, (ii) 156 mg/(g VSS) h under anaerobic condition, and (iii) 200 mg/(g VSS) h under aerobic conditions with energy also supplied from polyphosphate. A side stream, with initially anaerobic conditions for PHB accumulation and phosphorus release, and then aerobic conditions for PHB accumulation, was proposed. In this side stream, biomass with a high PHB content and a high PHB production rate could be both achieved.     

Biodegradation of 2-Chlorophenol by Rhodopseudomonas palustris

A phototrophic bacterial culture that assimilates 2-chlorophenol (2-CP) was enriched from effluents of paper and wood industry. The isolated bacterium was identified as Rhodopseudomonas palustris based on its morphological characteristics, biochemical reactions, and fatty acid methyl ester gas chromatography (FAME-GC) analysis. The bacterium R. palustris being photoheterotrophic in nutrition was also able to switch between phototropic, aerobic, and anaerobic modes of metabolism, as evidenced by modulation of the bacterial photopigments. The switching of anaerobic to aerobic metabolism was evidenced by the presence of catechol, a product of aerobic oxidation of 2-CP in the spent medium and the disappearance of photopigment-specific absorbance in the organism. R. palustris degraded about 97% of the supplemented 2-CP from the culture medium in 40 days along with production of an exo-polysaccharide, which probably provides protection from substrate toxicity and enhances its bioavailability. Thin-layer chromatography (TLC) and gas chromoatography–mass spectrometry (GC-MS) analysis of the R. palustris spent medium extracts indicated the presence of phenol. The GC-MS data also indicated that phenol is metabolized through an ortho-cleavage pathway. Further analysis of the R. palustris cell-free extracts for enzymes showed the presence of a chlorophenol dehalogenase (CD) and a chlorophenol nicotinamide adenine dinucleotide phosphate (NADPH)-oxidoreductase (CNOR) with respective specific activities of 0.114 and 0.26 μmol/min/mg, suggesting an initial reductive dechlorination step during 2-CP catabolism. The study thus highlights the important roles of phototrophic bacteria in the decontamination of environmental pollutants from the polluted sites.     

Phosphorus composition and release in sediment bacteria of the genus Pseudomonas during aerobic and anaerobic conditions

A substantial amount of sediment phosphorus can be bound in bacterial biomass. In this study the fractional composition of phosphorus in the bacteria Pseudomonas was determined by sequential extraction with ammonium chloride, sodium hydroxide and hydrochloric acid according to the scheme of Hieltjes & Lijklema (1980). Both non-labelled and ³²P-labelled bacteria were used for fractionation. Up to 80% of the bacterial phosphorus was found in the NaOH-nRP fraction, which is in agreement with the results of Hupfer & Uhlman (1992) for Acinetobacter and activated sludge obtained with the sequential extraction scheme of Psenner et al. (1985). A significant correlation was found between bacterial biomass and the amount of phosphorus retained in the NaOH-nRP fraction when sediments were fractionated. Additional experiments with ³²P-labelled Pseudomonas in sediment-water systems were performed in order to follow bacterial release of phosphorus under aerobic and anaerobic conditions. These studies did not sustain the hypothesis that anaerobic conditions lead to rapid release of phosphorus from bacterial cells.     

Radiolabelled proteomics to determine differential functioning of Accumulibacter during the anaerobic and aerobic phases of a bioreactor operating for enhanced biological phosphorus removal

Proteins synthesized by the mixed microbial community of two sequencing batch reactors run for enhanced biological phosphorus removal (EBPR) during aerobic and anaerobic reactor phases were compared, using mass spectrometry‐based proteomics and radiolabelling. Both sludges were dominated by polyphosphate‐accumulating organisms belonging to Candidatis Accumulibacter and the majority of proteins identified matched closest to these bacteria. Enzymes from the Embden–Meyerhof–Parnas pathway were identified, suggesting this is the major glycolytic pathway for these Accumulibacter populations. Enhanced aerobic synthesis of glyoxylate cycle enzymes suggests this cycle is important during the aerobic phase of EBPR. In one sludge, several TCA cycle enzymes showed enhanced aerobic synthesis, suggesting this cycle is unimportant anaerobically. The second sludge showed enhanced synthesis of TCA cycle enzymes under anaerobic conditions, suggesting full or partial TCA cycle operation anaerobically. A phylogenetic analysis of Accumulibacter polyphosphate kinase genes from each sludge demonstrated different Accumulibacter populations dominated the two sludges. Thus, TCA cycle activity differences may be due to Accumulibacter strain differences. The major fatty acids present in Accumulibacter‐dominated sludge include palmitic, hexadecenoic and cis‐vaccenic acid and fatty acid content increased by approximately 20% during the anaerobic phase. We hypothesize that this is associated with increased anaerobic phospholipid membrane biosynthesis, to accommodate intracellular polyhydroxyalkanoate granules.     

A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called ‘Candidatus Accumulibacter phosphatis’ was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge ‘biography’ be well understood – i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked. This revised version was published online in August 2006 with corrections to the Cover Date.     

A method for the selective enumeration and isolation of ruminal Lactobacillus and Streptococcus

Ruminal lactic acid-producing bacteria were selectively isolated and enumerated using a one hour aerobic exposure prior to incubation on a semi-selective Lactobacillus medium, MRS, under anaerobic conditions. The technique allowed growth of pure cultures of ruminal Lactobacillus spp. and Streptococcus bovis without supporting the growth of pure cultures of any of the prominent ruminal bacterial species. In mixed cultures, the one hour aerobic pre-incubation inhibited the growth of the obligate anaerobic ruminal bacteria which can otherwise grow on the MRS medium, and the subsequent anaerobic incubation permitted maximal recovery of the weakly aerotolerant ruminal lactic acid-producing Lactobacillus spp. and Streptococcus spp. The efficacy of this technique in selecting exclusively for the lactic acid-producing bacteria was also demonstrated from populations of rumen bacteria from mixed culture end-point in vitro fermentation, continuous in vitro culture and isolations from fresh ruminal samples.     

Isolation of a new type of polyphosphate accumulating bacterium and its phosphate removal characteristics

Polyphosphate accumulating bacteria were isolated from activated sludge cultured in a fill and draw system with alternating anaerobic and aerobic conditions. One of the isolates, strain NM-1 tentatively identified with the genus Micrococcus accumulated a large amount of phosphorus and its content reached 166 mg of phosphorus per g of cells. This strain accumulated phosphorus rapidly under aerobic conditions in the absence of any organic substrates, and when glucose was supplied to this strain under anaerobic conditions, it released phosphate and took up glucose in a similar manner to activated sludge that has good phosphorus eliminating capacity. Micro-aerophilic growth conditions were required for the induction of phosphorus accumulating activity.     

Aerobic and anaerobic de-epoxydation of mycotoxin deoxynivalenol by bacteria originating from agricultural soil

One hundred and fifty soil samples collected from different crop fields in southern Ontario, Canada were screened to obtain microorganisms capable of transforming deoxynivalenol (DON) to de-epoxy DON (dE-DON). Microbial DON to dE-DON transformation (i.e. de-epoxydation) was monitored by using liquid chromatography-ultraviolet-mass spectrometry (LC-UV–MS). The effects of growth substrates, temperature, pH, incubation time and aerobic versus anaerobic conditions on the ability of the microbes to de-epoxydize DON were evaluated. A mixed microbial culture from one composite soil sample showed 100% DON to dE-DON biotransformation in mineral salts broth (MSB) after 144 h of incubation. Treatments of the culture with selective antibiotics followed an elevated temperature (50°C) for 1.5 h considerably reduced the microbial diversity. Partial 16S-rRNA gene sequence analysis of the bacteria in the enriched culture indicated the presence of at least six bacterial genera, namely Serratia, Clostridium, Citrobacter, Enterococcus, Stenotrophomonas and Streptomyces. The enriched culture completely de-epoxydized DON after 60 h of incubation. Bacterial de-epoxydation of DON occurred at pH 6.0–7.5, and a wide array of temperatures (12–40°C). The culture showed rapid de-epoxydation activity under aerobic conditions compared to anaerobic conditions. This is the first report on microbial DON to dE-DON transformation under aerobic conditions and moderate temperatures. The culture could be used to detoxify DON contaminated feed and might be a potential source for gene(s) for DON de-epoxydation.     

A Remark on Microorganisms in Lake Sediments with Emphasis on Polyphosphate-accumulating Bacteria

Bacteria which are able to store large amounts of phosphorus as polyphosphate (PP) under aerobic conditions and to release it under anaerobic conditions are utilized for the advanced treatment of sewage. This note presents some results which indicate that PP-accumulating bacteria which possibly participate in the immobilization and release of dissolved phosphate may also be present in lake sediments.     

Characterization of diverse heterocyclic amine-degrading denitrifying bacteria from various environments

Although, there have been many published bacterial strains aerobically degrading the heterocyclic amine compounds, only one strain to date has been reported to degrade pyrrolidine under denitrifying conditions. In this study, denitrifying bacteria degrading pyrrolidine and piperidine were isolated from diverse geological and ecological origins through selective enrichment procedures. Based on the comparative sequence results of 16S rRNA genes, 30 heterocyclic amine-degrading isolates were grouped into ten distinct phylotypes belonging to the genera Thauera, Castellaniella, Rhizobium, or Paracoccus of the phylum Proteobacteria. The representative isolates of individual phylotypes were characterized by phylogenetic, phenotypic and chemotaxonomical traits, and dissimilatory nitrite reductase gene (nirK and nirS). All isolates completely degraded pyrrolidine and piperidine under both aerobic and anaerobic conditions. The anaerobic degradations were coupled to nitrate reduction. A metabolic pathway for the anaerobic degradation of pyrrolidine was proposed on the basis of enzyme activities implicated in pyrrolidine metabolism from three isolates. The three key pyrrolidine-metabolizing enzymes pyrrolidine dehydrogenase, γ-aminobutyrate/α-ketoglutarate aminotransferase, and succinic semialdehyde dehydrogenase, were induced by heterocyclic amines under denitrifying conditions. They were also induced in cells grown aerobically on heterocyclic amines, suggesting that the anaerobic degradation of pyrrolidine shares the pathway with aerobic degradation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of growth conditions of acetate utilization byRhodopseudomonas palustris isolated from a freshwater lake

Rhodopseudomonas palustris, a purple non-sulfur bacterium, was recently found throughout the water column in Lake Kinneret. It was demonstrated to be of a versatile nature, growing under both aerobic and anaerobic conditions at different light intensities. A comparison of C-acetate uptake byR. palustris andChlorobium phaeobacterioides, a green sulfur bacterium, showed that, under identical growth conditions, C-acetate assimilation byR. palustris was greater. Furthermore, C-acetate uptake forR. palustris was greater than C−CO₂ uptake at all light intensities. Depending on the prevailing conditions, acetate can be used byR. palustris as both an electron donor and carbon source. Malate synthase was used as an indicator of activity of the glyoxylic acid cycle. It was found that enzyme activity was higher (i.e., acetate was used mainly as a carbon source) under anaerobic conditions, in the dark, or in the absence of HCO ₃ ⁻ . Acetate was used preferably as an electron donor under photosynthetic microaerophillic conditions.     

Role of glycogen in acetate uptake and polyhydroxyalkanoate synthesis in anaerobic-aerobic activated sludge with a minimized polyphosphate content

The role of glycogen in the uptake of acetate in anaerobic-aerobic activated sludge without enhanced biological phosphorus removal were investigated. Although the polyphosphate content of the sludge was minimized by lowering the phosphorus feeding concentration, significant acetate uptake and accumulation of polyhydroxyalkanoates (PHAs) were observed in proportion to glycogen consumption under anaerobic conditions. The results of anaerobic inhibition studies, which showed suppressive effects on acetate uptake by a glycolysis inhibitor (iodoacetate) but not by a membrane ATPase inhibitor (N,N′-dicyclohexyl carbodiimide), supported an assumption that glycogen degradation through glycolysis supplies the required ATP and reducing power for PHA synthesis from acetate and consumed glycogen. Under subsequent aerobic conditions, the accumulated PHAs were depleted and the consumed glycogen recovered to the same level as that at the start of the anaerobic phase. Iodoacetate also inhibited the recovery of glycogen under aerobic conditions, suggesting that nearly 50% of the PHAs depleted was used for glycogen synthesis through reversed glycolysis.     

Atypical Polyphosphate Accumulation by the Denitrifying Bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-β-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

Biodegradation of azo dyes in cocultures of anaerobic granular sludge with aerobic aromatic amine degrading enrichment cultures

A prerequisite for the mineralization (complete biodegradation) of many azo dyes is a combination of reductive and oxidative steps. In this study, the biodegradation of two azo dyes, 4-phenylazophenol (4-PAP) and Mordant Yellow 10 (4-sulfophenylazo-salicylic acid; MY10), was evaluated in batch experiments where anaerobic and aerobic conditions were integrated by exposing anaerobic granular sludge to oxygen. Under these conditions, the azo dyes were reduced, resulting in a temporal accumulation of aromatic amines. 4-Aminophenol (4-AP) and aniline were detected from the reduction of 4-PAP. 5-Aminosalicylic acid (5-ASA) and sulfanilic acid (SA) were detected from the reduction of MY10. Subsequently, aniline was degraded further in the presence of oxygen by the facultative aerobic bacteria present in the anaerobic granular sludge. 5-ASA and SA were also degraded, if inocula from aerobic enrichment cultures were added to the batch experiments. Due to rapid autoxidation of 4-AP, no enrichment culture could be established for this compound. The results of this study indicate that aerobic enrichment cultures developed on aromatic amines combined with oxygen-tolerant anaerobic granular sludge can potentially be used to completely biodegrade azo dyes under integrated anaerobic/aerobic conditions. Received: 16 September 1998 / Received revision: 14 December 1998 / Accepted: 21 December 1998     

Stoichiometric model of the aerobic metabolism of the biological phosphorus removal process

In the aerobic phase of the biological phosphorus removal process, poly-beta-hydroxybutyrate, produced during anaerobic conditions, is used for cell growth, phosphate uptake, and glycogen formation. A metabolic model of this process has been developed. The yields for growth, polyphosphate and glycogen formation are quantified using the coupling of all these conversions to the oxygen consumption. The uptake of phosphate and storage as polyphosphate is shown to have a direct effect on the observed oxygen consumption in the aerobic phase. The overall energy requirements for the P-metabolism are substantial: 25% of the acetate consumed during anaerobic conditions and 60% of the oxygen consumptions is used for the synthesis of polyphosphate and glycogen. (c) 1994 John Wiley & Sons, Inc.     

Growth of Rhodopseudomonas palustris under phototrophic and non-phototrophic conditions and its CO-dependent H2 production

A new hydrogen producing bacterium, Rhodopseudomonas palustris P4, originally isolated under an anaerobic/phototrophic condition, grew well under aerobic/chemoheterotrophic or anaerobic/chemoheterotrophic conditions and showed CO-dependent, H₂ production activity when transferred to anaerobic conditions. Cell growth was best under an aerobic/chemoheterotrophic condition as the doubling time of 1 h, while the H₂ production activity was highest in the cells grown under an aerobic/chemoheterotrophic condition at 20 mmol g^–1 cell^–1 h^–1.     

Microbial transformation of chlorinated benzoates

Chlorinated benzoates enter the environment through their use as herbicides or as metabolites of other halogenated compounds. Ample evidence is available indicating biodegradation of chlorinated benzoates to CO₂ and chloride in the environment under aerobic as well as anaerobic conditions. Under aerobic conditions, lower chlorinated benzoates can serve as sole electron and carbon sources supporting growth of a large list of taxonomically diverse bacterial strains. These bacteria utilize a variety of pathways ranging from those involving an initial degradative attack by dioxygenases to those initiated by hydrolytic dehalogenases. In addition to monochlorinated benzoates, several bacterial strains have been isolated that can grow on dichloro-, and trichloro- isomers of chlorobenzoates. Some aerobic bacteria are capable of cometabolizing chlorinated benzoates with simple primary substrates such as benzoate. Under anaerobic conditions, chlorinated benzoates are subject to reductive dechlorination when suitable electron-donating substrates are available. Several halorespiring bacteria are known which can use chlorobenzoates as electron acceptors to support growth. For example, Desulfomonile tiedjei catalyzes the reductive dechlorination of 3-chlorobenzoate to benzoate. The benzoate skeleton is mineralized by other microorganisms in the anaerobic environment. Various dichloro- and trichlorobenzoates are also known to be dechlorinated in anaerobic sediments.     

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