DNA-based detection and characterisation of strictly anaerobic beer-spoilage bacteria

Megasphaera cerevisiae, Pectinatus cerevisiiphilus, Pectinatus frisingensis, Selenomonas lacticifex, Zymophilus paucivorans and Zymophilus raffinosivorans are strictly anaerobic Gram-stain-negative bacteria that are able to spoil beer by producing off-flavours and turbidity. They have only been isolated from the beer production chain. The species are phylogenetically affiliated to the Sporomusa sub-branch in the class "Clostridia". Routine cultivation methods for detection of strictly anaerobic bacteria in breweries are time-consuming and do not allow species identification. The main aim of this study was to utilise DNA-based techniques in order to improve detection and identification of the Sporomusa sub-branch beer-spoilage bacteria and to increase understanding of their biodiversity, evolution and natural sources. Practical PCR-based assays were developed for monitoring of M. cerevisiae, Pectinatus species and the group of Sporomusa sub-branch beer spoilers throughout the beer production process. The developed assays reliably differentiated the target bacteria from other brewery-related microbes. The contaminant detection in process samples (10 1,000 cfu/ml) could be accomplished in 2 8 h. Low levels of viable cells in finished beer (≤10 cfu/100 ml) were usually detected after 1 3 d culture enrichment. Time saving compared to cultivation methods was up to 6 d. Based on a polyphasic approach, this study revealed the existence of three new anaerobic spoilage species in the beer production chain, i.e. Megasphaera paucivorans, Megasphaera sueciensis and Pectinatus haikarae. The description of these species enabled establishment of phenotypic and DNA-based methods for their detection and identification. The 16S rRNA gene based phylogenetic analysis of the Sporomusa sub-branch showed that the genus Selenomonas originates from several ancestors and will require reclassification. Moreover, Z. paucivorans and Z. raffinosivorans were found to be in fact members of the genus Propionispira. This relationship implies that they were carried to breweries along with plant material. The brewery-related Megasphaera species formed a distinct sub-group that did not include any sequences from other sources, suggesting that M. cerevisiae, M. paucivorans and M. sueciensis may be uniquely adapted to the brewery ecosystem. M. cerevisiae was also shown to exhibit remarkable resistance against many brewery-related stress conditions. This may partly explain why it is a brewery contaminant. This study showed that DNA-based techniques provide useful tools for obtaining more rapid and specific information about the presence and identity of the strictly anaerobic spoilage bacteria in the beer production chain than is possible using cultivation methods. This should ensure financial benefits to the industry and better product quality to customers. In addition, DNA-based analyses provided new insight into the biodiversity as well as natural sources and relations of the Sporomusa sub-branch bacteria. The data can be exploited for taxonomic classification of these bacteria and for surveillance and control of contaminations.

Biodegradation of 1080: Testing soils in south-eastern Australia for sodium fluoroacetate-degrading micro-organisms

Sodium fluoroacetate (1080) is a vertebrate poison commonly used for the control of vertebrate pests in Australia. Long-term environmental persistence of 1080 from baiting operations has likely nontarget species and environmental impacts and is a matter of public concern. Defluorinating micro-organisms have been detected in soils of Western and central Australia, and Queensland, but not in south-eastern Australia. The presence or absence of defluorinating micro-organisms in soils from south-eastern Australia will assist in determining whether long-term environmental persistence of 1080 is or is not occurring. Soils from the Central West Slopes and Plains and Central Tablelands of New South Wales were sampled to investigate the presence and capability of 1080 defluorinating soil micro-organisms. Thirty-one species of micro-organisms were isolated from soils from each site after 10 days incubation in a 20 mM 1080 solution. Of these, 13 isolates showed measurable defluorinating ability when grown in a 1080 and sterile soil suspension. Two species, the bacteria Micromonospora, and the actinomycete Streptosporangium, have not been previously reported for their defluorinating ability. These results indicate that defluorinating micro-organisms are present in soils in south-eastern Australia, which adds weight to other studies that found that 1080 is subject to microbiological degradative processes following removal from the bait substrate. Soil micro-organism defluorination, in combination with physical breakdown and uptake by plants, indicates that fluoroacetate in soils and natural water ways is unlikely to persist. This has implications for the better informed use of 1080 in pest animal management programmes in south-eastern Australia.

Fate of pathogen indicators in a domestic blend of food waste and wastewater through a two-stage anaerobic digestion system

Anaerobic digestion is a viable on-site treatment technology for rich organic waste streams such as food waste and blackwater. In contrast to large-scale municipal wastewater treatment plants which are typically located away from the community, the effluent from any type of on-site system is a potential pathogenic hazard because of the intimacy of the system to the community. The native concentrations of the pathogen indicators Escherichia coli, Clostridium perfringens and somatic coliphage were tracked for 30 days under stable operation (organic loading rate (OLR) = 1.8 kgCOD m(-3) day(-1), methane yield = 52% on a chemical oxygen demand (COD) basis) of a two-stage laboratory-scale digester treating a mixture of food waste and blackwater. E. coli numbers were reduced by a factor of 10(6.4) in the thermophilic stage, from 10(7.5+/-0.3) to 10(1.1+/-0.1) cfu 100 mL(-1), but regenerated by a factor of 10(4) in the mesophilic stage. Neither the thermophilic nor mesophilic stages had any significant impact on C. perfringens concentrations. Coliphage concentrations were reduced by a factor of 10(1.4) across the two stages. The study shows that anaerobic digestion only reduces pathogen counts marginally but that counts in effluent samples could be readily reduced to below detection limits by filtration through a 0.22 microm membrane, to investigate membrane filtration as a possible sanitation technique.

Microbial activities in boreal soils: Biodegradation of organic contaminants at low temperature and ammonia oxidation

This thesis deals with the response of biodegradation of selected anthropogenic organic contaminants and natural autochthonous organic matter to low temperature in boreal surface soils. Furthermore, the thesis describes activity, diversity and population size of autotrophic ammonia-oxidizing bacteria (AOB) in a boreal soil used for landfarming of oil-refinery wastes, and presents a new approach, in which the particular AOB were enriched and cultivated in situ from the landfarming soil onto cation exchange membranes. This thesis demonstrates that rhizosphere fraction of natural forest humus soil and agricultural clay loam soil from Helsinki Metropolitan area were capable of degrading of low to moderate concentrations (0.2 50 µg cm-3) of PCP, phenanthrene and 2,4,5-TCP at temperatures realistic to boreal climate (-2.5 to +15 °C). At the low temperatures, the biodegradation of PCP, phenanthrene and 2,4,5-TCP was more effective (Q10-values from 1.6 to 7.6) in the rhizosphere fraction of the forest soil than in the agricultural soil. Q10-values of endogenous soil respiration (carbon dioxide evolution) and selected hydrolytic enzyme activities (acetate-esterase, butyrate-esterase and β-glucosidase) in acid coniferous forest soil were 1.6 to 2.8 at temperatures from -3 to +30 °C. The results indicated that the temperature dependence of decomposition of natural autochthonous soil organic matter in the studied coniferous forest was only moderate. The numbers of AOB in the landfarming (sandy clay loam) soil were determined with quantitative polymerase chain reaction (real-time PCR) and with Most Probable Number (MPN) methods, and potential ammonium oxidation activity was measured with the chlorate inhibition technique. The results indicated presence of large and active AOB populations in the heavily oil-contaminated and urea-fertilised landfarming soil. Assessment of the populations of AOB with denaturing gradient gel electrophoresis (DGGE) profiling and sequence analysis of PCR-amplified 16S rRNA genes showed that Nitrosospira-like AOB in clusters 2 and 3 were predominant in the oily landfarming soil. This observation was supported by fluorescence in situ hybridization (FISH) analysis of the AOB grown on the soil-incubated cation-exchange membranes. The results of this thesis expand the suggested importance of Nitrosospira-like AOB in terrestrial environments to include chronically oil-contaminated soils.

Functional genes and gene array analysis as tools for monitoring hydrocarbon biodegradation

Bioremediation, which is the exploitation of the intrinsic ability of environmental microbes to degrade and remove harmful compounds from nature, is considered to be an environmentally sustainable and cost-effective means for environmental clean-up. However, a comprehensive understanding of the biodegradation potential of microbial communities and their response to decontamination measures is required for the effective management of bioremediation processes. In this thesis, the potential to use hydrocarbon-degradative genes as indicators of aerobic hydrocarbon biodegradation was investigated. Small-scale functional gene macro- and microarrays targeting aliphatic, monoaromatic and low molecular weight polyaromatic hydrocarbon biodegradation were developed in order to simultaneously monitor the biodegradation of mixtures of hydrocarbons. The validity of the array analysis in monitoring hydrocarbon biodegradation was evaluated in microcosm studies and field-scale bioremediation processes by comparing the hybridization signal intensities to hydrocarbon mineralization, real-time polymerase chain reaction (PCR), dot blot hybridization and both chemical and microbiological monitoring data. The results obtained by real-time PCR, dot blot hybridization and gene array analysis were in good agreement with hydrocarbon biodegradation in laboratory-scale microcosms. Mineralization of several hydrocarbons could be monitored simultaneously using gene array analysis. In the field-scale bioremediation processes, the detection and enumeration of hydrocarbon-degradative genes provided important additional information for process optimization and design. In creosote-contaminated groundwater, gene array analysis demonstrated that the aerobic biodegradation potential that was present at the site, but restrained under the oxygen-limited conditions, could be successfully stimulated with aeration and nutrient infiltration. During ex situ bioremediation of diesel oil- and lubrication oil-contaminated soil, the functional gene array analysis revealed inefficient hydrocarbon biodegradation, caused by poor aeration during composting. The functional gene array specifically detected upper and lower biodegradation pathways required for complete mineralization of hydrocarbons. Bacteria representing 1 % of the microbial community could be detected without prior PCR amplification. Molecular biological monitoring methods based on functional genes provide powerful tools for the development of more efficient remediation processes. The parallel detection of several functional genes using functional gene array analysis is an especially promising tool for monitoring the biodegradation of mixtures of hydrocarbons.

Constitutive model for municipal solid waste incorporating mechanical creep and biodegradation-induced compression

A constitutive model is proposed to describe the stress-strain behavior of municipal solid waste (MSW) under loading using the critical state soil mechanics framework. The modified cam clay model is extended to incorporate the effects of mechanical creep and time dependent biodegradation to calculate total compression under loading. Model parameters are evaluated based on one-dimensional compression and triaxial consolidated undrained test series conducted on three types of MSW: (a) fresh MSW obtained from working phase of a landfill, (b) landfilled waste retrieved from a landfill after 1.5 years of degradation, and (c) synthetic MSW with controlled composition. The model captures the stress-strain and pore water pressure response of these three types of MSW adequately. The model is useful for assessing the deformation and stability of landfills and any post-closure development structures located on landfills.

Anaerobic DNA cleavage activity in red light and photocytotoxicity of (pyridine-2-thiol)cobalt(III) complexes of phenanthroline bases

Cobalt(III) complexes [Co(pnt)(B)(2)](NO3)(2) (1-3) of pyridine-2-thiol (pnt) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d: 2',3'-f]quinoxaline (dpq in 2) and dipyrido[3,2-a:2',3'-c] phenazine (dppz in 3), have been prepared, characterized and their photo-induced anaerobic DNA cleavage activity studied. The crystal structure of 1a as mixed ClO4- and PF6- salt of 1 shows a (CoN5S)-N-III coordination geometry in which the pnt and phen showed N,S- and N,N-donor binding modes, respectively. The complexes exhibit Co(III)/Co(II) redox couple near -0.3 V (vs. SCE) in 20% DMF-Tris-HCl buffer having 0.1 M TBAP. The complexes show binding propensity to calf thymus DNA giving K-b values within 2.2 x 10(4)-7.3 x 10(5) M-1. Thermal melting and viscosity data suggest DNA surface and/or groove binding of the complexes. The complexes show significant anaerobic DNA cleavage activity in red light under argon atmosphere possibly involving sulfide anion radical or thiyl radical species. The DNA cleavage reaction under aerobic medium in red light is found to involve both singlet oxygen and hydroxyl radical pathways. The dppz complex 3 shows non-specific BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via both hydroxyl and singlet oxygen pathways. The dppz complex 3 exhibits photocytotoxicity in HeLa cervical cancer cells giving IC50 values of 767 nM and 19.38 mu M in UV-A light of 365 nm and in the dark, respectively. A significant reduction of the dark toxicity of the dppz base (IC50 = 8.34 mu M in dark) is observed on binding to the cobalt(III) center.

Photocytotoxic and anaerobic DNA cleavage activity of binuclear 3,3 '-dithiodipropionic acid cobalt(II) complexes having phenanthroline bases

Dicobalt(II) complexes [{(B)Co-11)(2)(mu-dtdp)(2)] (1-3) of 3,3'-dithiodipropionic acid (dtdp) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq in 2) and dipyrido13,2-a:2',3'-clphenazine (dppz in 3), have been prepared, characterized and their photo-induced anaerobic DNA cleavage activity studied. The elemental analysis and mass spectral data suggest binuclear formulation of the complexes. The redox inactive complexes have magnetically non-interacting dicobalt(II) core showing magnetic moment of similar to 3.9 p per cobalt(II) center. The complexes show good binding propensity to calf thymus DNA giving K-b values within 4.3 x 10(5)-4.0 x 10(6) M-1. Thermal melting and viscosity data predict DNA groove binding and/or partial intercalative nature of the complexes. The complexes show significant anaerobic DNA cleavage activity in green light under argon atmosphere possibly involving radical species generated from the disulfide moiety in a type-I pathway. The DNA cleavage reaction under aerobic medium in green light is found to involve hydroxyl radical species. The dppz complex 3 exhibits significant photocytotoxicity in HeLa cervical cancer cells with an IC50 value of 2.31 mu M in UV-A light of 365 nm, while it is essentially non-toxic in dark giving an IC50 value of >200 mu M. A significant reduction of the dark toxicity of the organic dppz base (IC50 = 8.3 mu M in dark) is observed on binding to the cobalt(II) center while essentially retaining its photocytotoxicity in UV-A light (IC50 = 0.4 mu M). (C) 2010 Elsevier Ltd. All rights reserved.

Protoporphyrinogen IX oxidase from Plasmodium falciparum is anaerobic and is localized to the mitochondrion

Earlier studies in this laboratory had shown that the malarial parasite can synthesize heme de novo and inhibition of the pathway leads to death of the parasite. It has been proposed that the pathway for the biosynthesis of heme in Plasmodium falciparum is unique involving three different cellular compartments, namely mitochondrion, apicoplast and cytosol. Experimental evidences are now available for the functionality and localization of all the enzymes of this pathway, except protoporphyrinogen IX oxidase (PfPPO), the penultimate enzyme. In the present study. PfPPO has been cloned, expressed and shown to be localized to the mitochondrion by immunofluorescence microscopy. Interestingly, the enzyme has been found to be active only under anaerobic conditions and is dependent on electron transport chain (ETC) acceptors for its activity. The native enzyme present in the parasite is inhibited by the ETC inhibitors, atovaquone and antimycin. Atovaquone, a well known inhibitor of parasite dihydroorotate dehydrogenase, dependent on the ETC, inhibits synthesis of heme as well in P. falciparum culture. A model is proposed to explain the ETC dependence of both the pyrimidine and heme-biosynthetic pathways in P. falciparum. (C) 2010 Elsevier B.V. All rights reserved.

New pathway for the biodegradation of indole in Aspergillus niger

Indole and its derivatives form a class of toxic recalcitrant environmental pollutants. The growth of Aspergillus niger was inhibited by very low concentrations (0.005 to 0.02%) of indole, even when 125- to 500-fold excess glucose was present in the medium. When 0.02% indole was added, the fungus showed a lag phase for about 30 h and the uptake of glucose was inhibited. Indole was metabolized by a new pathway via indoxyl (3-hydroxyindole), N-formylanthranilic acid, anthranilic acid,2,3-dihydroxybenzoic acid, and catechol, which was further degraded by ortho cleavage. The enzymes N-formylanthranilate deformylase, anthranilate hydroxylase, 2,3-dihydroxybenzoate decarboxylase, and catechol dioxygenase were induced by indole as early as after 5 h of growth, and their activities were demonstrated in a cell-free system.

Anaerobic ammonium oxidation (anammox) in sediments of the Gulf of Finland

Anaerobic ammonium oxidation (anammox) and denitrification were measured in the open sea and coastal accumulation basins of the Gulf of Finland. The different methods used gave conflicting results on the importance of the anammox process in the sediments. Anammox generally contributed less than 20 % to the total N-2 production, and no anammox was found in a shallow inner estuary basin. However, the discovery of the anammox process in the open sea sediments challenges the denitrification measurements made in the area, as the coexistence of anammox and denitrification compromises the central assumptions behind the method used in denitrification measurements and causes overestimates of the N-2 production. The high (NO3-)-N-15 incubation concentration used in Baltic Sea denitrification measurements exacerbates this overestimation, which is likely to have been substantial.

Evidence for the involvement of multiple pathways in the biodegradation of 1- and 2-methylnaphthalene by pseudomonas putida CSV86

Pseudomonas putida CSV86, a soil bacterium, grows on 1- and 2-methylnaphthalene as the sole source of carbon and energy. In order to deduce the pathways for the biodegradation of 1- and 2-methylnaphthalene, metabolites were isolated from the spent medium and purified by thin layer chromatography. Emphasis has been placed on the structural characterisation of isolated intermediates by CC-MS, demonstration of enzyme activities in the cell free extracts and measurement of oxygen uptake by whole cells in the presence of various probable metabolic intermediates. The data obtained from such a study suggest the possibility of occurrence of multiple pathways in the degradation of 1- and 2-methylnaphthalene. We propose that, in one of the pathways, the aromatic ring adjacent to the one bearing the methyl moiety is oxidized leading to the formation of methylsalicylates and methylcatechols. In another pathway the methyl side chain is hydroxylated to -CH2-OH which is further converted to -CHO and -COOH resulting in the formation of naphthoic acid as the end product. In addition to this, 2-hydroxymethylnaphthalene formed by the hydroxylation of the methyl group of 2-methylnaphthalene undergoes aromatic ring hydroxylation. The resultant dihydrodiol is further oxidised by a series of enzyme catalysed reactions to form 4-hydroxymethyl catechol as the end product of the pathway.

Planar triazinium cations from VO2+-assisted ring cyclizations: a remarkably efficient thiazole species for nuclear staining, PDT and anaerobic photocleavage of DNA

Planar triazinium cationic species, from VO2+-assisted cyclization of 1-(2-thiazolylazo)-2-naphthol, shows efficient DNA intercalative binding, visible light-induced anaerobic plasmid DNA photocleavage activity and photocytotoxicity in HeLa and MCF-7 cancer cells by an apoptotic pathway with selective localization of the compound in the nucleus as evidenced from the nuclear staining and confocal imaging.

A pathway for biodegradation of 1-naphthoic acid by Pseudomonas maltophilia CSV89

Pseudomonas maltophilia CSV89, a bacterium isolated from soil in our laboratory, grows on 1-naphthoic acid as the sole source of carbon and energy. To elucidate the pathway for degradation of 1-naphthoic acid, the metabolites were isolated from spent medium, purified by TLC, and characterized by gas chromatography-mass spectrometry. The involvement of various metabolites as intermediates in the pathway was established by demonstrating relevant enzyme activities in cell-free extracts, oxygen uptake and transformation of metabolites by the whole cells. The results obtained from such studies suggest that the degradation of 1-naphthoic acid is initiated by double hydroxylation of the aromatic ring adjacent to the one bearing the carboxyl group, resulting in the formation of 1,2-dihydroxy-8-carboxynaphthalene. The resultant diol was oxidized via 3-formyl salicylate, 2-hydroxyisophthalate, salicylate and catechol to TCA cycle intermediates.