Biological control of problematic bacterial populations causing foaming in activated sludge wastewater treatment plants-phage therapy and beyond.

The production of a stable foam on the surfaces of reactors is a global operating problem in activated sludge plants. In many cases, these foams are stabilized by hydrophobic members of the Mycolata, a group of Actinobacteria whose outer membranes contain long-chain hydroxylated mycolic acids. There is currently no single strategy which works for all foams. One attractive approach is to use lytic bacteriophages specific for the foam stabilizing Mycolata population. Such phages are present in activated sludge mixed liquor and can be recovered readily from it. However, no phage has been recovered which lyses Gordonia amarae and Gordonia pseudoamarae, probably the most common foaming Mycolata members. Whole genome sequencing revealed that both G. amarae and G. pseudoamarae from plants around the world are particularly well endowed with genes encoding antiviral defence mechanisms. However, both these populations were lysed rapidly by a parasitic nanobacterium isolated from a plant in Australia. This organism, a member of the Saccharibacteria, was also effective against many other Mycolata, thus providing a potential agent for control of foams stabilized by them.

Isolation and characterization of bacteriophage SPI1, which infects the activated-sludge-foaming bacterium Skermania piniformis.

Foaming in activated sludge plants is a worldwide problem commonly caused by proliferation of bacteria of the order Corynebacteriales. These include Skermania piniformis, a filamentous bacterium that has been documented to be a major cause of foaming globally, and particularly in Australian treatment plants. Phage SPI1 is the first phage that was isolated and shown to infect this organism. It targets seven of the nine strains of S. piniformis held in our culture collection, but none of the other 73 mycolata strains of different genera, mostly isolated from wastewater, against which it was tested. Phage SPI1 is a member of the family Siphoviridae and has a circularly permuted dsDNA genome of 55,748 bp with a G+C content of 67.8 mol %. It appears to be obligatorily lytic, with no evidence of genes related to a lysogenic mode of existence.

The impact of horizontal gene transfer on targeting the internal transcribed spacer region (ITS) to identify Acinetobacter junii strains.

AIMS: Despite electrophoretic patterns of ITS PCR amplicons often suggesting only a single ITS sequence variant is present in strains of Acinetobacter junii, sequence data shows differences in ITS copies between and among them. This paper set out to explain why these ITS variants arise, and whether their presence compromises the reliability of the ITS targeted methods currently available for typing Ac. junii strains. METHODS AND RESULTS: ITS sequences from a number of strains of Ac. junii were either downloaded from public databases or generated here by cloning and sequencing ITS PCR amplicons. ITS copies of Ac. junii strain 97338 were all 666 bp long, with identical sequences. In Ac. junii ATCC 17908(T) /BCRC 14854(T) ), ITS copies were also all identical in their lengths but now were 706/7 bp long. Two sequence variants of these 707 bp ITS were detected. One was identical in its sequence to the nine ITS copies downloaded from the whole genome sequence of Ac. junii CIP 64·5, and those in several other Ac. junii strains. The other 707 bp ITS variant occurred elsewhere only in Ac. junii strain DSM 14968 of those examined. The six ITS copies from the genome sequence of Ac. junii NIPH 182 were all 685 bp, and with identical sequences. Ac. junii strain 178 also possessed this same 685 bp ITS variant, one of six variants detected there. At least five ITS sequence variants were seen in Ac. junii strain 97380, four in strain DSM 14968 and two in the whole genome of strain 107470. CONCLUSIONS: As with those of other Acinetobacter species, such ITS variants arise not from intragenomic recombination events but from the presence of different length indels. These arise from horizontal gene transfers involving ITS fragments of other Acinetobacter species. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of these indels compromises the reliability of the ITS targeted methods available for typing Acinetobacter junii. It also precludes the value of using ITS sequences as phylogenetic markers in members of the genus Acinetobacter, since the outcomes in both cases depends on which copy variant is chosen.

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans.

AIMS: To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans. METHODS AND RESULTS: In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0.13 g l(-1) N) initial NaNO(3) or (NH(4))(2)SO(4) levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0.78 g l(-1) N) initial NaNO(3) levels. EPS produced by CSTR grown cultures with high (NH(4))(2)SO(4) levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO(3) levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units. CONCLUSIONS: While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.

Ecophysiology of polyphosphate-accumulating organisms and glycogen-accumulating organisms in a continuously aerated enhanced biological phosphorus removal process.

AIMS: To investigate the ecophysiology of populations of polyphosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) in communities of a novel acetate fed process removing phosphate from wastewater. Attempts were made to see if acetate could be replaced by an alternative carbon source which did not support the growth of the GAO. METHODS AND RESULTS: A continuously aerated sequencing batch reactor was operated with different acetate feed levels. Fluorescence in situ hybridization (FISH) showed that Defluviicoccus GAO numbers increased at lower acetate feed levels. With FISH/microautoradiography (MAR) both detected morphotypes of Defluviicoccus assimilated a wider range of substrates aerobically than Accumulibacter PAO. Their uptake profile differed from that reported for the same phylotype in full scale anaerobic : aerobic EBPR plants. CONCLUSIONS: This suggests that replacing acetate with another substrate is unlikely to provide Accumulibacter with a selective advantage in this process. Why Defluviicoccus appeared to out-compete Accumulibacter at lower acetate concentrations was not clear. Data suggest physiological and morphological diversity may exist within a single Defluviicoccus phylotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This study implies that the current FISH probes for Defluviicoccus GAO may not reveal the full extent of their biodiversity, and that more information is required before strategies for their control can be devised.

Eikelboom filamentous morphotypes 0675 and 0041 embrace members of the Chloroflexi: resolving their phylogeny, and design of fluorescence in situ hybridisation probes for their identification.

Although the phylogeny of many of the filamentous bacteria responsible for bulking in activated sludge plants is now known, and fluorescence in situ hybridisation (FISH) probes have been designed for their in situ identification, there are some noticeable exceptions. This study reports the identification of the Eikelboom morphotypes 0041 and 0675. Because these morphotypes differ only in their filament diameters, they are often considered together in surveys based on microscopic identifications. Here we show that they are phylogenetically distinct, and so should be viewed no longer as morphological variants of a single population. Amplicon sequencing data of Australian EBPR plant biomass containing types 0041 and 0675, and phylogenetic analysis have revealed that both, like many other bulking filament morphotypes, are members of the phylum Chloroflexi and probably representatives of two different genera. FISH probes are described here targeting each. Surveys carried out on Australian activated sludge plants suggest that type 0675 occurs more in plants designed to remove phosphorus, while type 0041 shows no such preference, and was seen in biomass samples from a wide range of plant configurations.

Gene cassette-associated sequences from phosphorus and non-phosphorus removing microbial communities in aerobic:anaerobic sequencing batch reactors.

Mobile gene elements associated with integrons, including as gene cassettes, have been proposed to play an important role in bacterial evolution by providing an extensive genetic resource. This study hypothesized that critical genes for enzymes involved in EBPR systems, including those involved in polyphosphate, PHA and glycogen synthesis, may be present in mobile gene cassettes. Although no such genes were identified in any of the functional and deteriorated enhanced biological phosphorus removal (EBPR) laboratory-scale SBR systems examined here, many of the open reading frames (ORFs) remained unidentified because of the incompleteness of publicly available databases. An ORF of unknown function (SBR6-2) was encountered in deteriorated EBPR system with an unexpectedly high frequency, comprising 35% of the gene cassette-associated sequences for that system.

Substrate uptake by Gordonia amarae in activated sludge foams by FISH-MAR.

Gordonia amarae is a right-angled branching filament belonging to the mycolic acid-containing Actinobacteria which is commonly found in many foaming activated sludge wastewater treatment plants. Although studies on different substrates as sole carbon sources by pure cultures of G. amarae have been carried out, none have examined substrate uptake by this organism in situ. Uptake of several hydrophilic and hydrophobic substrates by G. amarae was evaluated in situ using a combination of fluorescence in situ hybridization and microautoradiography. G. amarae could assimilate a range of both hydrophilic and hydrophobic substrates. From the data, G. amarae appears to be physiologically active under aerobic, anaerobic and anoxic condition (NO2 and NO3) for some substrates. This might explain why attempts to control foaming caused by G. amarae using anoxic and anaerobic selectors have been unsuccessful. This study emphasizes that bacteria can behave differently in situ to pure cultures and that it is important to evaluate the in situ physiology of these bacteria if we are to better understand their role in the wastewater treatment process.

The in situ physiology of "Nostocoida limicola" II, a filamentous bacterial morphotype in bulking activated sludge, using fluorescence in situ hybridization and microautoradiography.

The in situ physiology of the actinobacterial bulking and foaming filamentous bacterium "Nostocoida limicola" II was studied by fluorescence in situ hybridization/microautoradiography. Substrate assimilation patterns of pure cultures of this bacterium were different to those seen in activated sludge biomass samples. There was no evidence to suggest that "N. limicola" II preferred hydrophobic substrates, but evidence was produced to support the view that it is metabolically active under anaerobic conditions in activated sludge.

Proteolytic inactivation of an extracellular (1-->3)-beta-glucanase from the fungus Acremonium persicinum is associated with growth at neutral or alkaline medium pH.

The filamentous fungus Acremonium persicinum released high levels of proteolytic enzyme activity into the culture fluid during growth at pH 7 or above. Almost total inhibition of this crude activity by phenylmethylsulfonyl fluoride suggested that it was mainly due to the presence of a serine protease. This protease inactivated one of three extracellular (1-->3)-beta-glucanases produced by this fungus, although the activities of the remaining two (1-->3)-beta-glucanases did not appear to be affected. Growth of A. persicinum in acidic conditions resulted in the presence of much lower extracellular proteolytic activity and no apparent (1-->3)-beta-glucanase inactivation.

Analysis of the structural diversity of mycolic acids of Rhodococcus and Gordonia [correction of Gordonla] isolates from activated sludge foams by selective ion monitoring gas chromatography-mass spectrometry (SIM GC-MS)

A method using Selective Ion Monitoring (SIM) gas chromatography-mass spectrometry is described for analysis of mycolic acids which reveals a hitherto unrecognised chemical structural diversity among these in some members of the Mycolata. The structural interpretation of mass spectral data of mycolic acids from Rhodococcus spp and Gordonia [corrected] spp using SIM is discussed.

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.

Effects of culture conditions on the mycolic acid composition of isolates of Rhodococcus spp. from activated sludgefoams.

The influence of two different carbon sources and three incubation temperatures on the mycolic acid compositions of three Rhodococcus isolates from activated sludge was examined using Selective Ion Monitoring (SIM) gas chromatography-mass spectrometry (GC-MS). Considerable qualitative and quantitative differences were detected in the mycolic acid compositions of the three very closely related isolates grown under the same conditions. Culture age also affected both the chain lengths and proportions of saturated mycolic acids detected in cell extracts, but not in the same manner for each isolate. Mycolic acids generally were of shorter chain lengths in cells grown with Tween 80 compared to glucose grown cells in strain 11R but the opposite situation occurred with strains A7 and D5. In all three, the proportion of unsaturated mycolic acids decreased with increasing growth temperatures. The taxonomic relevance of these observations and possible explanations for the observed changes in mycolic acid composition under various culture conditions are discussed.

Structure and functional analysis of the microbial community in an aerobic: anaerobic sequencing batch reactor (SBR) with no phosphorus removal.

The bacterial community of an aerobic:anaerobic non-P removing SBR biomass fed a mixture of acetate and glucose was analysed using several 16S rRNA based methods. Populations responsible for anaerobic glucose and acetate assimilation were determined with fluorescent in situ hybridization (FISH) in combination with microautoradiography (FISH/MAR). At 'steady state' this community consisted of alpha-Proteobacteria (26%) and gamma-Proteobacteria (14%), mainly appearing as large cocci in tetrads (i.e. typical 'G-Bacteria'). Large numbers of low G+C bacteria (22%), and high G+C Gram-positive bacteria (29%) seen as small cocci in clusters or in sheets were also detected after FISH. DGGE fingerprinting of PCR amplified 16S rDNA fragments and subsequent cloning and sequencing of several of the major bands led to the identification of some of these populations. They included an organism 98% similar in its 16S rRNA sequence to Micropruina glycogenica, and ca. 76% of the high G+C bacteria responded to a probe MIC 184, designed against it. The rest responded to the KSB 531 probe designed against a high G+C clone sequence, sbr-gs28 reported in other similar systems. FISH analyses showed that both these high G+C populations were almost totally dominated by small clustered cocci. Only ca. 2% of cells were beta-Proteobacteria. None of the alpha- and gamma-Proteobacterial 'G-bacteria' responded to FISH probes designed for the 'G-Bacteria' Amaricoccus spp. or Defluvicoccus vanus. FISH/MAR revealed that not all the alpha-Proteobacterial 'G-Bacteria' could take up acetate or glucose anaerobically. Almost all of the gamma-Proteobacterial 'G-Bacteria' assimilated acetate anaerobically but not glucose, the low G+C clustered cocci only took up glucose, whereas the high G+C bacteria including M. glycogenica and the sbr-gs28 clone assimilated both acetate and glucose. All bacteria other than the low G+C small cocci and a few of the alpha-Proteobacteria accumulated PHB. The low G+C bacteria showing anaerobic glucose assimilation ability were considered responsible for the lactic acid produced anaerobically by this SBR biomass, and M. glycogenica for its high glycogen content.

The filamentous bacterial morphotype 'Nostocoida limicola' I contains at least two previously described genera in the low G+C gram positive bacteria.

Isolates of eight bacterial filaments fitting the published morphological description of 'Nostocoida limicola' I were obtained from the mixed liquor of four different Australian and one Czech Republic activated sludge plants by micromanipulation. On the basis of their near complete (Ben 200 and Ben 201), or partial (Ben 77, Ben 78, Ben 202, Ben 203, Ben 204 and Ben 205) 16S rRNA gene sequences, six of these isolates were 99.3-100% similar to Lactosphaera pasteurii and Trichococcus flocculiformis, a bulking filament only reported previously in Germany. The other two (Ben 203 and Ben 204) were 99.9% similar to Streptococcus suis. Hence, all are in the low mol % G+C gram-positive bacteria division of the Bacteria. On this evidence 'N. limicola' I is phylogenetically unrelated to 'Nostocoida limicola' II, which is now known to be in the Actinobacteria, even though these two filamentous bacteria appearing in activated sludge systems have been considered to be closely related to each other historically.

Structure of epiglucan, a highly side-chain/branched (1 --> 3;1 --> 6)-beta-glucan from the micro fungus Epicoccum nigrum Ehrenb. ex Schlecht.

The extracellular fungal polysaccharide, epiglucan, synthesised by Epicoccum nigrum is a side-chain/branched (1 --> 3;1 --> 6)-D-beta-glucan. Methylation analysis, 13C DEPT NMR and specific enzymic digestion data show slight variation in branching frequency among the epiglucans from the three strains examined. The (1 --> 3)-beta-linked backbone has (1 --> 6)-beta-linked branches at frequencies greater than the homologous glucans, scleroglucan and schizophyllan, from Sclerotium spp. and Schizophyllum commune, respectively. The structural analyses do not allow a distinction to be made between structures I and II. [structures: see text] Epiglucan displays non-Newtonian shear thinning rheological properties, typical of these glucans.

Noncellulolytic fungal beta-glucanases: their physiology and regulation.

The occurrence, regulation, and action of fungal enzymes capable of degrading noncellulosic beta-glucans, especially 1,3-beta- and 1,6-beta-glucans, are reviewed. Special consideration is given to their roles in both metabolic and morphogenetic events in the fungal cell, including cell wall extension, hyphal branching, sporulation, budding, and autolysis. Also examined are the protocols currently available for their purification, with some of the properties of purified beta-glucanases discussed in terms of their potential applications in industrial, agricultural, and medical fields.

Purification and characterization of an extracellular beta-glucosidase from the filamentous fungus Acremonium persicinum and its probable role in beta-glucan degradation.

A beta-glucosidase from the culture filtrates of the filamentous fungus Acremonium persicinum has been purified by (NH4)2SO4 precipitation followed by anion-exchange and gel filtration chromatography. SDS-PAGE of the purified enzyme gave a single band with an apparent molecular mass of 128 kDa. The enzyme is a monomeric protein with an isoelectric point of 4.3 and a pH optimum of 5.5. Comparison of the N-terminal amino acid sequence revealed similarities between the A. persicinum enzyme and several other extracellular fungal beta-glucosidases including those from Trichoderma reesei, Aspergillus aculeatus, Saccharomycopsis fibuligera, and Pichia anomala. In addition to the hydrolysis of p-nitrophenyl-beta-glucoside, the enzyme was also active against several other aryl-beta-glucosides as well as a range of beta-linked oligoglucosides including laminaribiose, gentiobiose, cellobiose, and sophorose. D-Glucono-1,5-lactone and glucose are competitive inhibitors while the enzyme was also inhibited by N-bromosuccinimide, N-acetylimidazole, dicyclohexyl carbodiimide, Woodward's Reagent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4, and some metal ions. Possible roles for this enzyme in the noncellulolytic fungus A. persicinum are discussed in light of the increase in the rate of reducing sugar release from beta-glucans by (1-->3)- and (1-->6)-beta-glucanases when the beta-glucosidase is also present in the reaction mixtures.

Genomic fingerprinting of the 16S-23S gene spacer region suggests that novel Acinetobacter isolates are present in activated sludge.

The taxonomic status of the genus Acinetobacteris currently confused and the role of these organisms in activated sludge is poorly understood. Currently unidentified isolates of Acinetobacterfrom activated sludge were fingerprinted by making use of polymorphisms in their 16S-23S rDNA spacer region. The PCR amplified 16S-23S rDNA spacer region was digested with five different restriction enzymes to further differentiate between the isolates. The resulting band patterns were very diverse and the data suggests that the activated sludge isolates are different to the known genomic species of Acinetobacter which are predominantly clinical isolates. The results of this study imply the existence of yet unrecognised species of Acinetobacter in activated sludge.

Environmental factors contributing to the "G bacteria" population in full-scale EBPR plants.

A survey of several enhanced biological phosphorus removal (EBPR) plants within Australia has demonstrated that a group of bacteria known as the "G" bacteria are able to proliferate under a broad range of plant configurations. The diverse designs and operational parameters of these plants did not permit definitive determination of the factor(s) contributing to the proliferation of G bacteria. Two plants were monitored over time to assess the G bacteria and phosphorus accumulating organisms (PAO) populations in relation to key operational parameters. The mixed liquor biomass and operational parameters were compared to other plants successfully and unsuccessfully reducing phosphorus from the wastewater. Two critical factors recognised in this study were the dissolved oxygen concentration in the aerobic zone and the type and amount of carbon source in the anaerobic zone.