Analysis of the polyphosphate-accumulating microflora in phosphorus-eliminating, anaerobic-aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp.

Polyphosphate-accumulating gram-negative bacteria were isolated from different anaerobic-aerobic activated sludge systems with diverse processes for enhanced biological phosphorus (P) elimination. Of 22 isolates, 10 were allocated to the genus Acinetobacter by using multiple-test systems and soluble protein and polyamine patterns. As diaminopropane (DAP) appears to be the characteristic main polyamine compound produced by Acinetobacter spp., it was used as a biomarker for the genus. The high DAP contents of representative samples from municipal wastes with enhanced biological P elimination indicated that Acinetobacter spp. can be dominant organisms in sewage treatment plants with low organic loading and nitrification and denitrification steps. Contrary to accepted opinion, sludge from treatment plants with efficient P removal and high organic loading had a low DAP content, indicating that bacteria other than Acinetobacter spp. are responsible for enhanced biological P elimination in these plants.     

Biotechnology for phosphorus removal during wastewater treatment

Advanced biological wastewater treatment for the removal of phosphorus in excess of the normal metabolic requirements of activated sludge type processes has been developed as an alternative to chemical addition. Current laboratory and pilot plant investigations have confirmed that a preliminary anaerobic zone and plug-flow type configuration are necessary for good enhanced biological phosphorus removal. Nitrate in the anaerobic stage inhibits the process whereas acetate enhances phosphorus uptake. The bacteria probably responsible are of the Acinetobacter genus and the presence of stored polyphosphate within these bacteria has been demonstrated. It has also been shown that pure cultures of Acinetobacter do not necessarily take up soluble substrate as phosphate is released during the anaerobic phase, in contrast to the current proposed mechanism, and that in certain cases natural chemical precipitation could make a significant contribution towards overall phosphorus removal. Several studies of pilot and full-scale plants have been reported.     

Polyphosphate-accumulating bacteria from sewage plants with different proceses for biological phosphorus removal

Abstract Activated sludge samples from pilot plants using different processes for enhanced biological phosphorus removal were investigated for the occurrence of polyphosphate-accumulating bacteria. All samples showed a direct correlation between the relative number of phosphate-accumulating bacteria and phosphate uptake. Various species of bacteria with polyphosphate granules were found in sludge flocs, but in every case Acinetobacter -like cells formed part or the main part of the polyphosphate-accumulating bacterial population. The spectrum of the bacteria with stored polyphosphates varied, depending on the sewage composition on the one hand and on the processes used for phosphorus removal on the other hand.     

Indigenous bacteria with antagonistic and plant-growth-promoting activities improve slow-filtration efficiency in soilless cultivation

In tomato soilless culture, slow filtration allows one to control the development of diseases caused by pathogenic microorganisms. During the disinfecting process, microbial elimination is ensured by mechanical and biological factors. In this study, system efficacy was enhanced further to a biological activation of filter by inoculating the pozzolana grains contained in the filtering unit with 5 selected bacteria. Three strains identified as Pseudomonas putida and 2 as Bacillus cereus came from a filter whose high efficiency to eliminate pathogens has been proven over years. These 5 bacteria displayed either a plant growth promoting activity (P. putida strains) or antagonistic properties (B. cereus strains). Over the first months following their introduction in the filter, the bacterial colonisation of pozzolana grains was particularly high as compared to the one observed in the control filter. Conversely to Bacillus spp. populations, Pseudomonas spp. ones remained abundant throughout the whole cultural season. The biological activation of filter unit very significantly enhanced fungal elimination with respect to the one displayed by the control filter. Indeed, the 6-month period needed by the control filter to reach its best efficacy against Fusarium oxysporum was shortened for the bacteria-amended filter; in addition, a high efficacy filtration was got as soon as the first month. Fast colonization of pozzolana grains by selected bacteria and their subsequent interaction with F. oxysporum are likely responsible for filter efficiency. Our results suggest that Pseudomonas spp. act by competition for nutrients, and Bacillus spp. by antibiosis and (or) direct parasitism. Elimination of other fungal pathogens, i.e., Pythium spp., seems to differ from that of Fusarium since both filters demonstrated a high efficacy at the experiment start. Pythium spp. elimination appears to mainly rely on physical factors. It is worth noting that a certain percentage of the 5 pozzolana-inoculated bacteria failed to colonise the filter unit and were, thus, driven to the plants by the nutrient solution. Their contribution to the establishment of a beneficial microbial community in the rhizosphere is discussed.     

A fluorescently-labelled r-RNA targeted oligonucleotide probe for the in situ detection of G-bacteria of the genus Amaricoccus in activated sludge

A fluorescently-labelled r-RNAtargeted oligonucleotide probe specific for members of the genus Amaricoccus, which includes one group of the Gram-negative G-Bacteria seen in activated sludge systems, is described. These organisms, previously 'identified' on their distinctive morphology of cocci in tetrads, have been associated with poor performance of biological nutrient removal (EBNR) plants, by out-competing the polyphosphate accumulating bacteria. Methods of sample preparation for probing activated sludge are detailed, and preliminary surveys of 46 plants, using this probe, show that G-Bacteria belonging to the genus Amaricoccus are seen not only in large numbers in EBNR systems but also in conventional plants. The presence of single cells of this organism was common, emphasizing the dangers of relying on morphology and cell arrangement to identify these bacteria.     

Involvement of Acinetobacter sp. in the floc-formation in activated sludge process

The coaggregation behavior of Acinetobacter johnsonii S35 isolate with sewage bacteria was assessed by a spectrophotometric assay using different samples from a municipal wastewater treatment plant and a community plant. A. johnsonii S35 coaggregated well with other free bacteria and microflocs at the mixing ratios of 0.2:1-0.6:1 of A. johnsonii S35 and sewage samples. In addition, the size of coaggregates became larger (100 μm or more) under the same conditions. A. johnsonii S35 cells were highly adsorbed (adsorption=93-99%) onto sludge samples. Microbial adhesion to hydrocarbon (MATH) test and adsorption to octyl-Sepharose CL-4B showed that A. johnsonii S35 cells and sludge samples had a hydrophobic character. The population of Acinetobacter spp. in sewage treatment plants was 2-7% and its role in bioflocculation was discussed. The present study revealed that A. johnsonii S35 isolate can play as a bridging organism and contribute in floc-formation in activated sludge process.     

Effect of dissolved oxygen concentration on sludge settleability

This laboratory study presents a detailed evaluation of the effects of dissolved oxygen concentration and accumulation of storage polymers on sludge settleability in activated sludge systems with an aerobic selector. The oxygen and substrate availability regime were simulated in laboratory sequencing batch reactor systems. The experiments showed that low dissolved oxygen concentration (1.1 mg O₂ l^–1) had a strong negative effect on sludge settleability, leading to the proliferation of filamentous bacteria (Thiothrix spp., Type 021N and Type 1851). This negative effect was stronger at high chemical oxygen demand loading rate. This indicates that a compartmentalised (plug flow) aerobic contact tank, designed at short hydraulic residence time to guarantee a strong substrate gradient, with low dissolved oxygen concentration, might be worse for sludge settleability than an "overdesigned" completely mixed contact tank. Contrary to the general hypothesis, the maximum specific acetate uptake rate, poly--hydroxybutyrate production rate, and resistance to short starvation periods are similar in both poor- and well-settling sludge. The results of this study support our previous hypothesis on the importance of substrate gradients for the development of filamentous structures in biological flocs, from soluble organic substrate gradients to dissolved oxygen gradients in sludge flocs.     

Pyrolysis mass spectrometry (PyMS) and 16S-23S rDNA spacer region fingerprinting suggests the presence of novel acinetobacters in activated sludge

Screening of large numbers of Acinetobacter spp. from activated sludge systems with Pyrolysis Mass Spectrometry (PyMS) showed that many did not cluster tightly with the currently described genomic species which have been obtained mainly from clinical sources. Selected isolates were then genotypically fingerprinted using their 16S-23S rDNA spacer region, and again the data revealed considerable differences in the genomic fingerprints of many of these activated sludge isolates to the predominantly clinical genomic species. In fact, few could be identified from them. The possibility that the current speciation within this genus is not adequate to encompass all these environmental isolates is addressed in relation to the methods used to study the population dynamics of Acinetobacter in activated sludge.     

Monitoring precursor 16S rRNAs of Acinetobacter spp. in activated sludge wastewater treatment systems

Recently, Cangelosi and Brabant used oligonucleotide probes targeting the precursor 16S rRNA of Escherichia coli to demonstrate that the levels of precursor rRNA were more sensitive to changes in growth phase than the levels of total rRNA (G. A. Cangelosi and W. H. Brabant, J. Bacteriol. 179:4457-4463, 1997). In order to measure changes in the levels of precursor rRNA in activated sludge systems, we designed oligonucleotide probes targeting the 3' region of the precursor 16S rRNA of Acinetobacter spp. We used these probes to monitor changes in the level of precursor 16S rRNA during batch growth of Acinetobacter spp. in Luria-Bertani (LB) medium, filtered wastewater, and in lab- and full-scale wastewater treatment systems. Consistent with the previous reports for E. coli, results obtained with membrane hybridizations and fluorescence in situ hybridizations with Acinetobacter calcoaceticus grown in LB medium showed a more substantial and faster increase in precursor 16S rRNA levels compared to the increase in total 16S rRNA levels during exponential growth. Diluting an overnight culture of A. calcoaceticus grown in LB medium with filtered wastewater resulted in a pattern of precursor 16S rRNA levels that appeared to follow diauxic growth. In addition, fluorescence in situ hybridizations with oligonucleotide probes targeting total 16S rRNA and precursor 16S rRNA showed that individual cells of A. calcoaceticus expressed highly variable levels of precursor 16S rRNA when adapting from LB medium to filtered sewage. Precursor 16S rRNA levels of Acinetobacter spp. transiently increased when activated sludge was mixed with influent wastewater in lab- and full-scale wastewater treatment systems. These results suggest that Acinetobacter spp. experience a change in growth activity within wastewater treatment systems.     

Coaggregation among nonflocculating bacteria isolated from activated sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Evaluation of the environmental impact of microbial aerosols generated by wastewater treatment plants utilizing different aeration systems

Using three sampler devices (SAS, Andersen Six-Stages and All Glass Impinger), the environmental impact of bacterial and fungal aerosols generated by municipal wastewater treatment plants operating with different methods of sludge oxygenation were evaluated. The highest microbial concentrations were recovered above the tanks (2247 cfu m-3) and in downwind positions (1425 cfu m-3), where a linear correlation (P < 0.05) was found between the quantity of sewage treated and the entities of microbial aerosol dispersion. Moreover, an exponential increase (P < 0.05) in the bacteria recovered from the air occurred at increasing times of treatment. However, after long-term plant operation, high bacterial and fungal concentrations were found in almost all of the sites around the plant. Coliforms, enterococci, Escherichia coli and staphylococci were almost always recovered in downwind positions. Considerable fractions (20-40%) of sampled bacteria were able to penetrate the final stages of the Andersen apparatus and thus, are likely to be able to penetrate the lungs. The plant operating with a fine bubble diffused air system instead was found to generate rather low concentrations of bacteria and fungi; moreover, staphylococci and indicator micro-organisms were almost absent. Finally, salmonellae, Shigella, Pseudomonas aeruginosa and Aeromonas spp. were not detected in either of the plants. The results indicate a remarkable dispersion of airborne bacteria and fungi from tanks in which oxygen is supplied via a mechanical agitation of sludge, and suggest the need to convert them to diffused aeration systems which pose a lesser hazard for human health.     

Bacterial community dynamics in long‐term operation of a pilot plant using aerobic granular sludge to treat pig slurry

Aerobic granular sludge represents an interesting approach for simultaneous organic matter and nitrogen removal in wastewater treatment plants. However, the information about microbial communities in aerobic granular systems dealing with industrial wastewater like pig slurry is limited. Herein, bacterial diversity and dynamics were assessed in a pilot scale plant using aerobic granular sludge for organic matter and nitrogen elimination from swine slurry during more than 300 days. Results indicated that bacterial composition evolved throughout the operational period from flocculent activated sludge, used as inoculum, to mature aerobic granules. Bacterial diversity increased at the beginning of the granulation process and then declined due to the application of transient organic matter and nitrogen loads. The operational conditions of the pilot plant and the degree of granulation determined the microbial community of the aerobic granules. Brachymonas, Zoogloea and Thauera were attributed with structural function as they are able to produce extracellular polymeric substances to maintain the granular structure. Nitrogen removal was justified by partial nitrification (Nitrosomonas) and denitrification (Thauera and Zoogloea), while Comamonas was identified as the main organic matter oxidizing bacteria. Overall, clear links between bacterial dynamics and composition with process performance were found and will help to predict their biological functions in wastewater ecosystems improving the future control of the process. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1212–1221, 2016     

Sludge dewatering and stabilization in drying reed beds: Characterization of three full-scale systems in Catalonia,Spain

Optimization of sludge management can help reducing sludge handling costs in wastewater treatment plants. Sludge drying reed beds appear as a new and alternative technology which has low energy requirements, reduced operating and maintenance costs, and causes little environmental impact. The objective of this work was to evaluate the efficiency of three full-scale drying reed beds in terms of sludge dewatering, stabilization and hygienisation. Samples of influent sludge and sludge accumulated in the reed beds were analysed for pH, Electrical Conductivity, Total Solids (TS), Volatile Solids (VS), Chemical Oxygen Demand, Biochemical Oxygen Demand, nutrients (Total Kjeldahl Nitrogen (TKN) and Total Phosphorus (TP)), heavy metals and faecal bacteria indicators (Escherichiacoli and Salmonella spp.). Lixiviate samples were also collected. There was a systematic increase in the TS concentration from 1–3% in the influent to 20–30% in the beds, which fits in the range obtained with conventional dewatering technologies. Progressive organic matter removal and sludge stabilization in the beds was also observed (VS concentration decreased from 52–67% TS in the influent to 31–49% TS in the beds). Concentration of nutrients of the sludge accumulated in the beds was quite low (TKN 2–7% TS and TP 0.04–0.7% TS), and heavy metals remained below law threshold concentrations. Salmonella spp. was not detected in any of the samples, while E. coli concentration was generally lower than 460 MPN/g in the sludge accumulated in the beds. The studied systems demonstrated a good efficiency for sludge dewatering and stabilization in the context of small remote wastewater treatment plants.     

Salmonella isolated in sewage sludge traced back to human cases of salmonellosis

AIM: The main aim of this study was to investigate a possible connection between the Salmonella content in sewage sludge and human cases of salmonellosis. An additional aim was to survey the antimicrobial resistance situation in Salmonella isolated from Swedish sewage sludge. METHODS AND RESULTS: The Salmonella strains were compared by restriction enzyme analysis combined with pulsed field gel electrophoresis and antimicrobial susceptibility testing. This study suggests a link between Salmonella isolated from sewage sludge and human Salmonella isolates. CONCLUSIONS: This study demonstrates that Salmonella spp. isolated in sewage treatment plants (STP) originate from infected humans and survive treatment at STP. It also highlights the risk of spreading resistant Salmonella strains from sewage sludge to the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: As Salmonella spp. originating from infected humans can survive the treatment at STP, the risk of Salmonella spp. being spread with sewage sludge to the environment and then to people and animals is enhanced. The threat to society is even worse if the bacteria are resistant to antimicrobial agents.     

Microbial selection of polyphosphate-accumulating bacteria in activated sludge wastewater treatment processes for enhanced biological phosphate removal

Activated sludge processes with alternating anaerobic and aerobic conditions (the anaerobic-aerobic process) have been successfully used for enhanced biological phosphate removal (EBPR) from wastewater. It is known that polyphosphate-accumulating bacteria (PAB) play an essential role for EBPR in the anaerobic-aerobic process. The present paper reviews limited information available on the metabolism and the microbial community structure of EBPR, highlighting the microbial ecological selection of PAB in EBPR processes. Exposure of microorganisms to alternate carbon-rich anaerobic environments and carbon-poor aerobic environments in the anaerobic-aerobic process induces the key metabolic characteristics of PAB, which include organic substrate uptake followed by its conversion to stored polyhydroxyalkanoate (PHA) and hydrolysis of intracellular polyphosphate accompanied by subsequent Pi release under anaerobic conditions. Intracellular glycogen is assumed to function as a regulator of the redox balance in the cell. Storage of glycogen is a key strategy for PAB to maintain the redox balance in the anaerobic uptake of various organic substrates, and hence to win in the microbial selection. Acinetobacter spp., Microlunatus phosphovorus, Lampropedia spp., and the Rhodocyclus group have been reported as candidates of PAB. PAB may not be composed of a few limited genospecies, but involve phylogenetically and taxonomically diverse groups of bacteria. To define microbial community structure of EBPR processes, it is needed to look more closely into the occurrence and behavior of each species of PAB in various EBPR processes mainly by molecular methods because many of PAB seem to be impossible to culture.     

The filamentous morphotype Eikelboom Type 1863 is not a single genetic entity

Five isolates of a filamentous bacterial morphotype with the distinctive diagnostic microscopic features of Eikelboom Type 1863 were obtained from activated sludge sewage treatment plants in Victoria, Australia. On the basis of phenotypic evidence and 16S rDNA sequence data, these isolates proved to be polyphyletic. Two (Ben 06 and Ben 06C) are from the Chryseobacterium subgroup which is in the Cytophaga group, subdivision I of the Flexibacter – Cytophaga – Bacteroides phylum. Two (Ben 56 and Ben 59) belong to the genus Acinetobacter , and one (Ben 58) is a Moraxella sp., closest to Mor. osloensis . The significance of these findings to the reliance on microscopic features for identification of these filamentous bacteria in activated sludge is discussed.     

不动杆菌属(Acinetobacter)细菌降解石油烃的研究进展

不动杆菌属细菌分布广泛,作为重要的石油烃降解者,在乳化和降解石油烃、降低石油烃生物毒性等方面有重要作用。本文概述了不动杆菌属细菌对烷烃、芳香烃等石油烃组分的降解,总结了该属细菌中已发现的烷烃氧化酶和芳香烃氧化酶,综述了该属细菌所分泌的表面活性剂的类型和乳化机理,讨论了固定化对该属细菌降解石油烃的影响,展望了该属细菌降解石油烃的应用前景。基于此,作者认为探索不动杆菌属细菌降解石油烃的详细机理和途径、发现关键酶、寻找遗传工具、构建基因工程菌、发掘环境友好的固定化材料,应是未来的研究重点及热点。     

Polyphosphate-degrading enzymes in Acinetobacter spp. and activated sludge

Polyphosphate-degrading enzymes were studied in Acinetobacter spp. and activated sludge. Polyphosphate: AMP phosphotransferase activity in Acinetobacter strain 210A decreased with increasing growth rates. The activity of this enzyme in cell extracts of Acinetobacter strain 210A was maximal at a pH of 8.5 and a temperature of 40 degrees C and was stimulated by (NH4)2SO4. The Km for AMP was 0.6 mM, and the Vmax was 60 nmol/min per mg of protein. Cell extracts of this strain also contained polyphosphatase, which was able to degrade native polyphosphate and synthetic magnesium polyphosphate and was strongly stimulated by 300 to 400 mM NH4Cl. A positive correlation was found between polyphosphate:AMP phosphotransferase activity, adenylate kinase activity, and phosphorus accumulation in six Acinetobacter strains. Significant activities of polyphosphate kinase were detected only in strain P, which contained no polyphosphate:AMP phosphotransferase. In samples of activated sludge from different plants, the activity of adenylate kinase correlated well with the ability of the sludge to remove phosphate biologically from wastewater.     

Polyphosphate-degrading enzymes in Acinetobacter spp. and activated sludge.

Polyphosphate-degrading enzymes were studied in Acinetobacter spp. and activated sludge. Polyphosphate: AMP phosphotransferase activity in Acinetobacter strain 210A decreased with increasing growth rates. The activity of this enzyme in cell extracts of Acinetobacter strain 210A was maximal at a pH of 8.5 and a temperature of 40 degrees C and was stimulated by (NH4)2SO4. The Km for AMP was 0.6 mM, and the Vmax was 60 nmol/min per mg of protein. Cell extracts of this strain also contained polyphosphatase, which was able to degrade native polyphosphate and synthetic magnesium polyphosphate and was strongly stimulated by 300 to 400 mM NH4Cl. A positive correlation was found between polyphosphate:AMP phosphotransferase activity, adenylate kinase activity, and phosphorus accumulation in six Acinetobacter strains. Significant activities of polyphosphate kinase were detected only in strain P, which contained no polyphosphate:AMP phosphotransferase. In samples of activated sludge from different plants, the activity of adenylate kinase correlated well with the ability of the sludge to remove phosphate biologically from wastewater.     

Isolation and characterization of filamentous bacteria present in bulking activated sludge

Summary Several types of filamentous microorganisms were observed and identified in samples of poorly settling (bulking) activated sludge. The major types encountered and the frequency (percentage) of appearance in the total of all treatment plants sampled were: Eikelboom type 0041 (60), type 1701 (45), Haliscomenobacter hydrossis (35), type 021N (30), Thiothrix spp. (20), and Sphaerotilus natans (20). Isolation techniques and culture media were developed and used to recover 42 axenic strains of filamentous bacteria from sludge samples collected. The isolates were identified as strains of Thiothrix, Beggiatoa, S. natans, and Eikelboom types 021N, 1701, 0041, and 0803. Nutritional and differential characterization of the bacteria was important to the differentiation of groups which could not be easily distinguished on the basis of morphology. Although certain treatment plant operating parameters (organic loading) seemed associated with the presence of specific filamentous organism types, possible interaction among factors precluded definite establishment of a cause and effect relationship for most of the treatment plant characteristics and organisms observed.