Phylogenetic and physiological characterization of organic waste-degrading bacterial communities

One of the greatest challenges in contemporary society is to reduce and treat household solid waste. The choice of inoculum to be used for start-up in reactors that degrade organic waste is critical to the success of organic waste treatment. In this study, the functional diversity, phylogenetic identification, and biogas production of bacterial communities from six inoculum sources were investigated. We used BIOLOG EcoPlates to evaluate the metabolic abilities of the bacterial communities, followed 16S rRNA gene sequence analysis to determine the phylogenetic affiliation of the bacteria responsible for carbon consumption. We observed great diversity in the physiological profiles. Of the six inocula tested, the sludge from an upflow anaerobic sludge blanket reactor (SRU) contained the most diverse, metabolically versatile microbiota and was characterized by the highest level of biogas production. By contrast, the sludge of the anaerobic lagoon (SAL) showed the worst performance in BIOLOG EcoPlates assays, but it exhibited the most diversity and generated the second largest amount of biogas. The bacterial isolates retrieved from BIOLOG EcoPlates were characterized as aerobic and/or facultative anaerobic, and were mainly Gram-negative. Phylogenetic analysis revealed that the isolates belonged to three major phyla: Proteobacteria, Firmicutes and Actinobacteria, represented by 33 genera. Proteobacteria exhibited the most diversity. The distribution of the bacterial genera differed considerably among the six inocula. Pseudomonas and Bacillus, which are able to degrade a wide range of proteins and carbohydrates, predominated in five of the six inocula. Analysis of the bacterial communities in this study indicates that both SRU and SAL microbiota are candidates for start-up inocula in anaerobic reactors. These start-up inocula must be studied further in order to identify their practical applications in degrading organic waste.     

Growth ofZymomonas mobilis CP4 on mannitol

Summary The ethanologenZymomonas mobilis has a restricted substrate range, namely glucose, fructose and sucrose. It would be useful to expand its substrate range to include other carbohydrates.Z. mobilis was screened for growth on 30 different carbohydrates and organic acids. A single spontaneous mutant,Z mobilis CP4.60, was isolated which illustrated feeble growth on mannitol as the sole carbohydrate source after three months of incubation. Growth ofZ. mobilis CP4.60 for several months in continuous culture with excess mannitol, and including a round of NTG (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis in the chemostat, led to the isolation a sequential series of mutants (CP4.62, CP4.64 and CP4.66), each with improved growth rates on mannitol. Metabolism of mannitol byZ. mobilis is oxygen-dependent, resulting in limited production of ethanol and incresed production of lactic acid. This is an initial example of extension of the substrate range ofZymomonas. The conversion of mannitol to fructose could be via an altered alcohol dehydrogenase.     

Prevalence of co-resistance to disinfectants and clinically relevant antibiotics in bacterial isolates from three hospital laboratory wastewaters in Southwestern Nigeria

The prevalence of co-resistance to four disinfectants and seven antibiotics was investigated among 57 bacterial strains isolated from the effluents of three hospital laboratories in Ogbomoso, Southwestern Nigeria. The organisms belonging to seven genera of public health importance such as Pseudomonas, Streptococcus, Serratia, Staphylococcus, Klebsiella, Proteus and Bacillus showed varying degrees of resistance to the test antimicrobial agents ranging from 0% to 77.8%. From among 25 organisms isolated from hospital A were recognized 16 phenotypic patterns of co-resistance to the test disinfectants and antibiotics; while from hospitals B and C were recognized 13 and 9 patterns, respectively, from among 18 and 14 isolates. The observed co-resistance to antimicrobial agents among the organisms reported in the present study is an indication of the risks posed by the untreated effluents to public health. It also adds to the increasing evidence about the role of hospital wastewaters as environmental reservoir of multi-drug-resistant bacteria.     

Performance evaluation of mixed and pure microbial inocula as surrogate culture in a BOD5 test

The use of activated sludge (AS) to assess the environmental impact of chemicals and wastewaters suffers from several drawbacks related to the heterogeneity, absence of standardization and health risk associated with such a mixed sewage population. To search for reliable testing inoculum alternatives, the potential of a standardized soil inoculum and a pure culture (Pseudomonas sp.), as reference material in a 5-day BOD test, was evaluated using the dilution and manometric methods and testing the standard glucose-glutamic acid solution. The results obtained for the BOD₅ of the standard solution fall in the range referred for the AS in the standards of the cited methods, demonstrating the potential of these inocula as an alternative to AS in the BOD₅ test. Moreover, testing on real wastewater samples showed highly significant correlations (P < 0.001) between the BOD₅ values obtained with these two inocula and with the standard inoculum. Analysis of metabolic patterns also pointed to a similar catabolic profile between soil inoculum and AS and to the potential of a mixed Pseudomonas-soil inoculum as an AS surrogate culture.     

The diversity of extradiol dioxygenase (edo) genes in cresol degrading rhodococci from a creosote-contaminated site that express a wide range of degradative abilities

Analysis of the bacterial population of soil surface samples from a creosote-contaminated site showed that up to 50% of the culturable micro-organisms detected were able to utilise a mixture of cresols. From fifty different microbial isolates fourteen that could utilise more than one cresol isomer were selected and identified by 16S rRNA analysis. Eight isolates were Rhodococcus strains and six were Pseudomonas strains. In general, the Rhodococcus strains exhibited a broader growth substrate range than the Pseudomonas strains. The distribution of various extradiol dioxygenase (edo) genes, previously associated with aromatic compound degradation in rhodococci, was determined for the Rhodococcus strains by PCR detection and Southern-blot hybridization. One strain, Rhodococcus sp. I1 exhibited the broadest growth substrate range and possessed five different edogenes. Gene disruption experiments indicated that two genes (edoC and edoD) were associated with isopropylbenzene and naphthalene catabolism respectively. The other Rhodococcus strains also possessed some of the edo genes and one (edoB) was present in all of the Rhodococcus strains analysed. None of the rhodococcal edo genes analysed were present in the Pseudomonas strains isolated from the site. It was concluded that individual strains of Rhodococcus possess a wide degradative ability and may be very important in the degradation of complex mixtures of substrates found in creosote.     

Substrate specificities of the chloromuconate cycloisomerases from Pseudomonas sp. B13, Ralstonia eutropha JMP134 and Pseudomonas sp. P51

The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted cis,cis-muconates. 2,4-Dichloro-cis,cis-muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro-cis,cis-muconate was the highest, while the constants for cis,cis-muconate, 2-chloro- and 2,5-dichloro-cis,cis-muconate were especially poor. The sequence of ClcB of the 3-chlorobenzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different from the published sequences). Corresponding to 2-chloro-cis,cis-muconate being a major metabolite of 3-chlorobenzoate degradation, the k _cat/K _m with 2-chloro-cis,cis-muconate was relatively high, while that with the still preferred substrate 2,4-dichloro-cis,cis-muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes. TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred substrate. Received: 7 August 1998 / Received revision: 24 November 1998 / Accepted: 29 November 1998     

The Effects of Cyanobacterial Exudates on Bacterial Growth and Biodegradation of Organic Contaminants

The pulp and paper industry largely depends on the biodegradation activities of heterotrophic bacteria to remove organic contaminants in wastewater prior to discharge. Our recent discovery of extensive cyanobacterial communities in pulp and paper waste treatment systems led us to investigate the potential impacts of cyanobacterial exudates on growth and biodegradation efficiency of three bacterial heterotrophs. Each of the three assessed bacteria represented different taxa commonly found in pulp and paper waste treatment systems: a fluorescent Pseudomonad, an Ancylobacter aquaticus strain, and a Ralstonia eutropha strain. They were capable of utilizing phenol, dichloroacetate (DCA), or 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. Exudates from all 12 cyanobacterial strains studied supported the growth of each bacterial strain to varying degrees. Maximum biomass of two bacterial strains positively correlated with the total organic carbon content of exudate treatments. The combined availability of exudate and a known growth substrate (i.e., phenol, DCA, or 2,4-D) generally had a synergistic affect on the growth of the Ancylobacter strain, whereas mixed effects were seen on the other two strains. Exudates from four representative cyanobacterial strains were assessed for their impacts on phenol and DCA biodegradation by the Pseudomonas and Ancylobacter strains, respectively. Exudates from three of the four cyanobacterial taxa repressed phenol biodegradation, but enhanced DCA biodegradation. These dissimilar impacts of cyanobacterial exudates on bacterial degradation of contaminants suggest a species-specific association, as well as a significant role for cyanobacteria during the biological treatment of wastewaters.     

Dechlorination of high-molecular-mass compounds in spent sulphite bleach effluents by free and immobilized cells of streptomycetes

Streptomyces chromofuscus and S. rochei dechlorinated high-molecular-mass compounds (HMM) from industrial bleach effluents. Compounds of the effluents from the first chlorination [(C + D)_red] and the subsequent alkaline extraction stage (E₁O) of a sulphite cellulose pulp mill were used as substrates for the microbial transformations. HMM bleach effluent fractions obtained by ultrafiltration were treated by free and immobilized cells in two types of bioreactors. The dechlorination was followed by measuring the reduction of activated-carbon-adsorbable organic-bound halogen (AOX) and the release of inorganic chloride. While 38–45% of the organic bound chlorine was released from a mixture of (C + D)_red and E₁O stage effluents within 20 days of incubation with S. chromofuscus, only 11–16% were liberated from E₁O-stage HMM bleach-effluent compounds by S. chromofuscus and S. rochei. Immobilized cells of both strains remained active for over 100 days in successive batches, each releasing 12–24% of chloride from HMM bleach-effluent compounds.     

Selecting inocula for the biodegradation of organic compounds at low concentrations

The inability of many organisms to degrade pollutants at low concentrations is a problem when selecting inocula for bioremediation of sites with these low concentrations. Thus, a study was conducted to determine the effect of low concentrations of p-nitrophenol (PNP) on growth of four PNP-degrading bacteria and their abilities to metabolize low concentrations of the compound in culture and samples from an oligotrophic lake. PNP did not increase the growth rates of Flavobacterium sp. M4, Pseudomonas sp. K, Flavobacterium sp. M1, and Pseudomonas sp. SP3 at concentrations of less than 2, 4, 10, and 100 ng/ml, respectively, when it was the sole added carbon source in culture, but it stimulated multiplication at higher concentrations. In liquid culture with the nitro compound as sole added carbon source, the four bacteria extensively mineralized PNP at 50 and 100 ng/ml, and three of the four degraded much of the substrate at 25 ng/ml. Pseudomonas sp. SP3 mineralized more than 20% but the two Flavobacterium strains converted less than 10% of the substrate to C02 at 10 ng/ml, and none of the three mineralized more than 5% at 1 and 5 ng PNP/ml. Under conditions where more than 99% of the radioactivity from ¹⁴C-PNP added at 1 ng/ml remained in solution, two of the isolates formed organic products. Pseudomonas sp. K had no activity at 1, 5, and 10 ng/ml. In contrast, when each of the bacteria was separately inoculated into samples of water from an oligotrophic lake and from a well in which PNP was not biodegraded, the bacteria were able to mineralize as little as 1 ng PNP/ml. The addition to a salts solution of 10 ng of glucose per ml resulted in mineralization of PNP at concentrations too low to be mineralized when the nitro compound was the sole source of added carbon. Bacteria may thus be able to mineralize substrates in natural waters at concentrations below those suggested by tests conducted in culture media, possibly because of the availability of other carbon sources for the bacteria.Offprint requests to: M. Alexander.     

Biodegradation of 4-chlorobenzoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PA01 NC

A bacterial consortium capable of degrading chloroaromatic compounds was isolated from pulp and paper mill effluents by selective enrichment on 4-chlorobenzoic acid as sole source of carbon and energy. The four different bacterial isolates obtained from bacterial consortium were identified as Pseudomonas aeruginosa AY792969 (A), P. aeruginosa PA01 NC (B), Pseudomonas sp. ZZ5 DQ113452 (C) and Pseudomonas sp. AY762360 (D) based on their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. These bacterial isolates were found to be versatile in degrading a variety of chloroaromatic compounds including fluoro- and iodobenzoic acids. P. aeruginosa PA01 NC utilized 4-chlorobenzoic acid at 2 g/l as growth substrate. Biodegradation studies have revealed that this organism degraded 4-chlorobenzoic acid through 4-chlorocatechol which was further metabolized by ortho-cleavage pathway and the dechlorination occurred after the ring-cleavage.     

Diverse cellular colonizing endophytic bacteria in field shoots and in vitro cultured papaya with physiological and functional implications

The study was envisaged to assess the extent of normally uncultivable endophytic bacteria in field papaya plants and in vitro established cultures adopting cultivation vs molecular analysis and microscopy. Surface‐sterilized axillary shoot‐buds of papaya ‘Arka Surya’ revealed high bacterial diversity as per 16S rRNA metagene amplicon sequencing (6 phyla, 10 classes, 21 families) with an abundance of Pseudomonas (Gammaproteobacteria), which also formed a common contaminant for in vitro cultured field explants. Molecular analysis of seedling shoot‐tip‐derived healthy proliferating cultures of three genotypes (‘Arka Surya’, ‘Arka Prabhath’, ‘Red Lady’) with regular monthly subculturing also displayed high bacterial diversity (11–16 phyla, >25 classes, >50 families, >200 genera) about 12–18 months after initial establishment. ‘Arka Surya’ and ‘Red Lady’ cultures bore predominantly Actinobacteria (75–78%) while ‘Arka Prabhath’ showed largely Alphaproteobacteria corroborating the slowly activated Methylobacterium sp. Bright‐field direct microscopy on tissue sections and tissue homogenate and epi‐fluorescence microscopy employing bacterial DNA probe SYTO‐9 revealed abundant intracellular bacteria embracing the next‐generation sequencing elucidated high taxonomic diversity. Phylogenetic investigation of communities by reconstruction of unobserved states‐ PICRUSt‐ functional annotation suggested significant operational roles for the bacterial‐biome. Metabolism, environmental information processing, and genetic information processing constituted major Kyoto Encyclopedia of Genes and Genomes KEGG attributes. Papaya stocks occasionally displayed bacterial growth on culture medium arising from the activation of originally uncultivable organisms to cultivation. The organisms included Bacillus (35%), Methylobacterium (15%), Pseudomonas (10%) and seven other genera (40%). This study reveals a hidden world of diverse and abundant conventionally uncultivable cellular‐colonizing endophytic bacteria in field shoots and micropropagating papaya stocks with high genotypic similarity and silent participation in various plant processes/pathways.     

Pressure enhances thermal stability of DNA polymerase from three thermophilic organisms

DNA polymerases derived from three thermophilic microorganisms, Pyrococcus strain ES4, Pyrococcus furiosus, and Thermus aquaticus, were stabilized in vitro by hydrostatic pressure at denaturing temperatures of 111°C, 107.5°C, and 100°C (respectively). Inactivation rates, as determined by enzyme activity measurements, were measured at 3, 45, and 89 MPa. Half-lives of P. strain ES4, P. furiosus, and T. aquaticus DNA polymerases increased from 5.0, 6.9, and 5.2 minutes (respectively) at 3 MPa to 12, 36, and 13 minutes (respectively) at 45 MPa. A pressure of 89 MPa further increased the half-lives of P. strain ES4 and T. aquaticus DNA polymerases to 26 and 39 minutes, while the half-life of P. furiosus DNA polymerase did not increase significantly from that at 45 MPa. The decay constant for P. strain ES4 and T. aquaticus polymerases decreased exponentially with increasing pressure, reflecting an observed change in volume for enzyme inactivation of 61 and 73 cm³/mol, respectively. Stabilization by pressure may result from pressure effects on thermal unfolding or pressure retardation of unimolecular inactivation of the unfolded state. Regardless of the mechanism, pressure stabilization of proteins could explain the previously observed extension of the maximum temperature for survival of P. strain ES4 and increase the survival of thermophiles in thermally variable deep-sea environments such as hydrothermal vents. Received: September 12, 1997 / Accepted: February 24, 1998     

A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase and α-galactosidase

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase A (XylA) from Neocallimastix patriciarum in combination with mannanase and α-galactosidase. Mannanase A (ManA) from Pseudomonas fluorescens subsp. cellulosa and ManA from Clostridium thermocellum, both family 26 glycosyl hydrolases, are structurally diverse and exhibit different pH and temperature optima. Although neither mannanase was effective in pretreating softwood pulp alone, both enzymes were able to enhance the production of reducing sugar and the reduction of single-stage bleached κ number when used with the xylanase. Sequential incubations with XylA and P. fluorescens ManA produced the largest final κ number reduction in comparison to control pretreated pulp. The release of galactose from softwood pulp by α-galactosidase A (AgaA) from P. fluorescens was enhanced by the presence of ManA from the same microorganism, and a single pretreatment with these enzymes, in combination with XylA, gave the most effective κ number reduction using a single incubation. Results indicated that mixtures of hemicellulase activities can be chosen to enhance pulp bleachability. Received: 16 August 1999 / Received revision: 13 December 1999 / Accepted: 15 December 1999     

Effects of organic compounds on the degradation ofp-nitrophenol in lake and industrial wastewater by inoculated bacteria

Many microorganisms fail to degrade pollutants when introduced in different natural environments. This is a problem in selecting inocula for bioremediation of polluted sites. Thus, a study was conducted to determine the success of four inoculants to degradep-nitrophenol (PNP) in lake and industrial wastewater and the effects of organic compounds on the degradation of high and low concentrations of PNP in these environments.Corynebacterium strain Z4 when inoculated into the lake and wastewater samples containing 20 µg/ml of PNP degraded 90% of PNP in one day. Addition of 100 µg/ml of glucose as a second substrate did not enhance the degradation of PNP and the bacterium utilized the two substrates simultaneously. Glucose used at the same concentration (100 µg/ml), inhibited degradation of 20 µg of PNP in wastewater byPseudomonas strain MS. However, glucose increased the extent of degradation of PNP byPseudomonas strain GR. Phenol also enhanced the degradation of PNP in wastewater byPseudomonas strain GR, but had no effect on the degradation of PNP byCorynebacterium strain Z4.Addition of 100 µg/ml of glucose as a second substrate into the lake water samples containing low concentration of PNP (26 ng/ml) enhanced the degradation of PNP and the growth ofCorynebacterium strain Z4. In the presence of glucose, it grew from 2×10⁴ to 4×10⁴ cells/ml in 3 days and degraded 70% of PNP as compared to samples without glucose in which the bacterium declined in cell number from 2×10⁴ to 8×10³ cells/ml and degraded only 30% PNP. The results suggest that in inoculation to enhance biodegradation, depending on the inoculant, second organic substrate many play an important role in controlling the rate and extent of biodegradation of organic compounds.Abbreviations PNP p-nitrophenol     

The predation on Asellus aquaticus (L.) by perch,Perca fluviatilis (L.), in a small forest lake

Asellus aquaticus (L.) was the most important benthic food item for perch, Perca fluviatilis (L.), in a small, extremely humic forest lake in southern Finland. The proportion of A. aquaticus in the diet of perch varied according to the former's availability, which, in turn, depended on its life cycle. Perch 11.0–12.9 cm (total length) most frequently fed on A. aquaticus; smaller and larger perch fed more frequently on crustacean zooplankton and aquatic insect larvae, respectively. The high density of the perch population, the importance of A. aquaticus in the benthos of the study lake and the general high activity level of the prey resulted in a high predation (0.1–1.8% per day). The population of A. aquaticus was also limited by the scarcity of macrophytic vegetation and the small area of oxygenated littoral in the lake.     

Efficiency of Root Respiration of a Flood-Tolerant and a Flood-Intolerant Senecio Species as Affected by Low Oxygen Tension

A method was described to determine root growth respiration and root maintenance respiration rate of plants, grown in culture solutions of high and low oxygen concentrations, during linear growth. Root growth respiration of aerobically grown plants was three to four times lower in the flood-intolerant Senecio jacobaea L. than in the flood-tolerant species. Senecio aquaticus Hill. Root growth respiration of Senecio aquaticus Hill decreased by a factor two to three upon transplantation to a culture solution of low oxygen tension. The difference in root growth respiration between aerobically and anaerobically grown Senecio aquaticus Hill was ascribed to the presence of a highly active non-phosphorylating oxidase.     

Isolation and preliminary characterization of a Zymomonas mobilis mutant with an altered preference for xylose and glucose utilization

The narrow substrate range of Zymomonas mobilis CP4 has been extended previously to include metabolism of the pentose sugar, xylose, by Zhang et al. (Science 267: 240–243). The strain CP4(pZB5) co-ferments both glucose and xylose in mixed sugar fermentations, however glucose is utilized preferentially. The present work reports the isolation of a new mutant from CP4(pZB5) which displays an altered carbon substrate preference. The mutant, CP4(pZB5) M1-2, metabolizes xylose more rapidly than glucose in mixed glucose/xylose media. Sequence data analysis revealed mutations in both the glucose facilitator (glf) and glucokinase (glk) genes.     

Endophytic bacteria enhancing growth and disease resistance of potato (Solanum tuberosum L.)

Priming plants by non-pathogenic bacteria allows the host to save energy and to reduce time needed for development of defense reaction during a pathogen attack. However, information on the role of endophytes in plant defense is limited. Here, the ability of endophytic bacteria to promote growth and resistance of potato plants towards infection by the necrotroph Pectobacterium atrosepticum was studied. A Pseudomonas sp. strain was selected due to antagonism towards bacterial pathogens and a Methylobacterium sp. strain because of efficient plant colonization. The aim of this study was to find if there is any correlation between plant growth promotion and induction of resistance by endophytes of potato, as well as to study the putative mechanisms of endophytes interacting with the plant during resistance induction. Both tested strains promoted growth of potato shoots but only the Pseudomonas sp. increased potato resistance towards the soft rot disease. Induction of disease resistance by the Methylobacterium sp. was inversely proportional to the size of bacterial population used for inoculation. The plant antioxidant system was moderately activated during the induction of resistance by the biocontrol strains. qPCR data on expression of marker genes of induced systemic resistance and acquired systemic resistance in endophyte-infected Arabidopsis plants showed activation of both salicylic acid and jasmonate/ethylene-dependent pathways after challenge inoculation with the pathogen. We suggest that some endophytes have the potential to activate both basal and inducible plant defense systems, whereas the growth promotion by biocontrol strains may not correlate with induction of disease resistance.