Cultural and biochemical diversity of pink-pigmented bacteria isolated from paper mill slimes

A study of 25 paper mill slime deposits and one additive revealed nine pink-pigmented bacterial isolates, eight of which were different from pink-pigmented bacteria identified in the paper industry in the middle 1900s. The pink-pigmented bacteria described previously in pulp and paper included Micrococcus agilis, Bacillus subtilis, Serratia sp. and Alcaligenes viscosus. With the exception of one isolate, Micrococcus sp., these isolates possessed many cultural, biochemical and chemical properties which were different from the ones previously reported for paper mills. Eight of these bacteria were Gram-negative rods or filamentous, aerobic and positive for catalase production. Two isolates were methylotrophic, oxidizing methanol and identified as Methylobacterium zatmanii. Cellular fatty acid analysis and other characteristics showed one isolate to be Roseomonas sp. Using 16S rRNA gene sequencing, one isolate which was a Gram-negative rod was identified as Deionococcus grandis. Four bacteria had cells that were long or filamentous and these were isolated from mills with pink slime problems. The identity of one of the filamentous bacteria was determined by 16S rRNA gene sequencing to be close to Flectobacillus sp. strain MWH38. Most of the isolates were susceptible to 11 industrial biocides. Journal of Industrial Microbiology & Biotechnology (2000) 25, 74–80. Received 28 January 2000/ Accepted in revised form 09 June 2000     

Degradation of pentachlorophenol by <Emphasis Type="Italic">Kocuria</Emphasis> sp. CL2 isolated from secondary sludge of pulp and paper mill

A pentachlorophenol (PCP) degrading bacterium was isolated and characterized from sludge of pulp and paper mill. This isolate used PCP as its sole source of carbon and energy and was capable of degrading this compound, as indicated by stoichiometric release of chloride and biomass formation. Based on morphology, biochemical tests, and 16S rRNA gene sequence analysis this strain was identified as Kocuria sp. CL2. High Performance Liquid Chromatography (HPLC) analysis revealed that this strain was able to degrade PCP up to a concentration of 600 mg/l. This is first time we are reporting the degradation of PCP by the Kocuria species. This isolate was also able to remove 58.64% of PCP from the sludge within two weeks. This study showed that the removal efficiency of PCP by CL2 was found to be very effective and can be used in degradation of PCP containing pulp paper mill waste in the environment.     

Biosorption of Copper (II) by the Microorganisms Isolated from the Crude-Oil-Contaminated Soil

The objective of this study was to isolate and screen the highly efficient copper-removing microorganisms from the petroleum hydrocarbon (PH)-contaminated sites in the Amazonian rain forest in Ecuador. Two bacterial strains (strain UEAB3 and UEAB6) have shown 100% microbial resistance on the nutrient medium containing 100 mM of MgCl₂, FeCl₃, and Al₂(SO₄)₃ separately. Though these two strains were less tolerant of ZnCl₂ and CuSO₄.5H₂O, they have proven 100% resistance at the lower concentrations of these two metals. According to atomic absorption spectroscopy (AAS) analysis, the filamentous fungi (strains UEAFr and UEAFg) were significantly (p<0.05) effective at bacteria in the biosorption (97–100%) of copper (5 mg L^?1) over 7 d. As per 16/18S rDNA sequences, UEAB3, UEAB6, UEAFr, and UEAFg were Bacillus thuringiensis, Bacillus cereus, Geomyces pannorum, and Geomyces sp., respectively. From these results, it can be comprehensively concluded that the isolated microbial cultures had a capacity to remove the copper metal from the liquid medium.     

Production of Laccase and Optimization of Its Production by Bacillus sp. Using Distillery Spent Wash as Inducer

A bacillus sp. isolated from the sediments of a distillery mill was used for laccase production under optimized culture conditions. The distillery effluent was used as an inducer for overproduction of laccase by employing the Taguchi approach. Screening of different medium components and their effect on laccase production was studied using an M-16 orthogonal array. The formation of laccase was considerably increased by addition of 1 mM copper sulfate (51.95 U/ml), which was further enhanced by the use of different inducers. The usefulness of the Taguchi method for optimization of culture conditions was investigated with five selected factors at four levels, and it was observed that the optimized medium resulted in a 9-fold increase in extracellular laccase production compared with the control. The optimized medium composition for laccase production was dextrose (1%), tryptone (0.1%), CuSO₄ (1 mM), and an inducer (distillery effluent 10% [v/v]) at pH 7, which altogether resulted in 107.32 U/ml extracellular laccase activity. Hence, the Taguchi approach proved to be a reliable tool in optimizing culture conditions and achieving the best possible combination for enhanced laccase production.     

Laboratory studies on biomachining of copper using Staphylococcus sp.

The possibility of using bacteria to drill metallic surfaces has been demonstrated using Staphylococcus sp., a facultative anaerobic bacterium, isolated from corroded copper piping. The experiment involved exposure of copper coupons (25 mm × 15 mm × 3 mm) to a culture of Staphylococcus sp. for a maximum period of 7 days. Coupons exposed to sterile bacterial growth medium were used as controls. Exposed coupons were removed intermittently and observed microscopically for the extent of drilling. The total pit area and volume on these coupons were determined using image analysis. The results showed that both the biomachined area and volume increased with the duration of coupon exposure. In the drilling experiment, a copper thin film 2 μm thick was perforated by this bacterium within a period of 7 days. In conclusion, the results suggested that bacteria can be used as a tool for machining metallic surfaces.     

Genomic Analysis and Comparative Hexavalent Chromium Reduction Potential of Predominant Bacillus species Isolated from Chromite Mine Soil

Ten predominant bacteria (CSB 1-10), isolated from chromite mine soil of Sukinda, Odisha, were characterized by means of biochemical and 16S rRNA gene sequencing. All of the bacterial isolates were Gram-positive, spore-forming, and motile rods with diameter ranging from 1.57–2.79 μm and identified as Bacillus species. Based on 16S rRNA gene sequencing and phylogenetic tree construction, 10 Bacillus species were grouped into two clusters: Bacillus subtilis cluster with four species (two of B. amyloliquefaciens, and one each of B. tequilensis and B. mojavensis); and Bacillus cereus cluster containing six species of B. cereus. Secondary rRNA structure predicted for all 10 bacteria using 16S rRNA sequences revealed some degree of genetic variations among the species. Among the isolated bacteria, CSB-9 was found to be the most efficient chromate reducing strain (0.8 × 10^?4 mg mg^?1 h^?1) in comparison to the others. Chromate reduction of the bacteria was associated with the contribution of extracellular enzyme production, and highest enzyme activity (0.9 ± 0.09 U mL^?1) was observed in CSB-9 (B. amyloliquefaciens). The present study revealed the dominance of Bacillus species in the chromite mine soil and their potential for bioremediation of hexavalent chromium from the polluted environments.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Evaluation of biosorption potency of <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. for removal of hexavalent chromium from tannery effluent

Two bacterial consortia were developed by continuous enrichment of microbial population of tannery and pulp and paper mill effluent contained Serratia mercascens, Pseudomonas fluorescence, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter sp. identified by 16S rDNA method. The consortia evaluated for removal of chromate [(Cr(VI)] in shake flask culture indicated pulp and paper mill consortium had more potential for removal of chromate. Acinetobacter sp. isolated from pulp and paper mill consortium removed higher amount of chromate [Cr(VI)] under aerobic conditions. Parameters optimized in different carbon, nitrogen sources, and pH, indicated maximum removal of chromate in sodium acetate (0.2%), sodium nitrate (0.1%) and pH 7 by Acinetobacter sp. Bacteria was applied in 2-l bioreactor significantly removed chromate after 3 days. The results of the study indicated removal of more than 75% chromium by Acinetobacter sp. determined by diphenylcarbazide colorimetric assay and atomic absorption spectrophotometer after 7 days. Study of microbial [Cr(VI)] removal and identification of reduction intermediates has been hindered by the lack of analytical techniques. Therefore, removal of chromium was further substantiated by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) which indicated bioaccumulation of chromium in the bacterial cells.     

Fermentation of methoxyacetate to glycolate and acetate by newly isolated strains of Acetobacterium sp.

Three strains of new mesophilic homoacetogenic bacteria were enriched and isolated from sewage sludge and from marine sediment samples with methoxyacetate as sole organic substrate in a carbonate-buffered medium under anoxic conditions. Two freshwater isolates were motile, Gram-positive, non-sporeforming rods. The marine strain was an immotile, Gram-positive rod with a slime capsula. All strains utilized only the methyl residue of methoxyacetate and released glycolic acid. They also fermented methyl groups of methoxylated aromatic compounds and of betaine to acetate with growth yields of 6–10 g dry matter per mol methyl group. H₂/CO₂, formate, methanol, hexamethylene tetramine, as well as fructose, numerous organic acids, glycerol, ethylene glycol, and glycol ethers were fermented to acetate as well. High activities of carbon monoxide dehydrogenase (0.4–2.2 U x mg protein^–1) were detected in all three isolates. The guanine-plus-cytosine-content of the DNA of the freshwater isolates was 42.7 and 44.4 mol %, with the marine isolate it was 47.7 mol %. The freshwater strains were assigned to the genus Acetobacterium as new strains of the species A. carbinolicum. One freshwater isolate, strain KoMac1, was deposited with the Deutsche Sammlung von Mikroorganismen GmbH, Braunschweig, under the number DSM 5193.     

Biosorption of Long Half-life Radionuclide of Strontium Ion (Sr+) by Marine Actinobacterium Nocardiopsis sp. 13H

Microorganisms may have a direct action on the fate of organic/inorganic ions in the environment through biosorption, detoxification or bioaccumulation processes. In the present study, the marine actinobacterial Nocardiopsis sp. 13H strain, isolated from different nuclear power plant (NPP) sites, India, tested for long half-life radionuclide of strontium (Sr⁺) was studied. Nocardiopsis sp. 13H could remove 93.6 ± 0.8% of Sr⁺ from test solutions containing 10 mM SrCl₂. The biosorption of Sr⁺ was studied for optimum condition of pH, temperature and time interval of remediation. Further, Sr⁺ adsorption was confirmed by scanning electron microscopy (SEM) coupled with energy-dispersive spectroscopy (EDS). In addition, with the presence of Sr⁺, extracellular polymeric substances (EPSs) were also found more and suggested interaction of EPS surface active assemblages being involved in radionuclide remediation. Fourier transform infrared (FTIR) study confirmed that Nocardiopsis sp. 13H cell surfaces are involved in Sr⁺ adsorption with the presence of dominant functional groups such as carboxyl, hydroxyl and amide groups. The results conclude that marine actinobacterial Nocardiopsis sp. 13H could be useful for the treatment of long half-life radioisotope pollution as an eco-friendly biosorbent in marine environmental cleanup.     

Heliobacterium sulfidophilum sp. nov. andHeliobacterium undosum sp. nov.: Sulfideoxidizing heliobacteria from thermal sulfidic springs

Two new species of heliobacteria isolated from cyanobacterial mats of two alkaline sulfidic hot springs are formally described. Strains BR4 and BG29 are assigned to anoxygenic phototrophic bacteria of the familyHeliobacteriaceae, since they possess the unique properties of this taxon: strict anaerobiosis, formation of bacteriochlorophyllg, the lack of extensive intracytoplasmic membranes and chlorosomes, an unusual cell wall structure, and phylogenetic relatedness to the low G+C gram-positive eubacteria. Based on the 16S rDNA sequence similarity, strains BR4 and BG29 are assigned to the genusHeliobacterium and described as two new species of this genus:Heliobacterium sulfidophilum sp. nov. andHeliobacterium undosum sp. nov. The G+C content of the DNA is 51.3 mol % inHbt. sulfidophilum and 57.2-57.7 mol % inHbt. undosum. The cells ofHbt. sulfidophilum are rods, and the cells ofHbt. undosum are slightly twisted spirilla or short rods. Both new bacteria are motile by peritrichous flagella.Hbt. sulfidophilum produces endospores. The new bacteria are strict anaerobes growing photoheterotrophically on a limited range of organic compounds. In the dark, they can switch from photosynthesis to the slow fermentation of pyruvate. Biotin is required as a growth factor. Both species are highly tolerant to sulfide (up to 2 mM at pH 7.5) and oxidize it photoheterotrophically to elemental sulfur; photoautotrophic growth was not observed. The temperature optimal for growth ofHbt. sulfidophilum andHbt undosum is 30–35‡C, and the optimal pH is 7–8.     

Morphological and pathogenic variability of Indian isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt

Wilt of chickpea (Cicer arietinum) caused by Fusarium oxysporum f. sp. ciceris is prevalent in almost all chickpea growing areas of the world and its incidence varied from 14.1 to 32.0% in the different states of India surveyed. The isolates were highly variable in their colony growth pattern, size of colony and pigmentations. The size of microconidia varied from 5.1–12.8 × 2.5–5.0 μm, whereas macroconidia ranged from 16.5–37.9 × 4.0–5.9 μm with 1–5 septations. One hundred and twelve isolates were grouped into 12 categories on the basis of their radial growth, size of macroconidia and growth pattern. Majority of the isolates were highly pathogenic causing more than 50% wilt in chickpea cultivar JG 62. Virulence analysis of 56 representative isolates on a set of 18 cultivars of chickpea, including 10 international differentials, grouped them into three categories. Chickpea cultivar KWR 108 differentiated all isolates of Punjab, Haryana and Delhi states and a few isolates of Rajasthan from others by showing resistant reactions and were placed in the first group. The rest of the isolates of Rajasthan state showed susceptible reactions on KWR 108 placed in a second group. Cultivar CPS 1 distinguished the isolates of Jharkhand state and placed them into a third group. An international set of cultivars recommended for race differentiation were not able to distinguish all the isolates into known races of the pathogen, therefore cultivar KWR 108 should be included in the existing differential set of cultivars.     

Phylogenetic and physiological characterization of mesophilic and thermophilic bacteria from a sewage sludge composting process in Sapporo,Japan

The phylogenetic and physiological characteristics of mesophilic and thermophilic bacteria isolated from a field-scale sewage sludge composter were determined by 16S rDNA and phenotype analyses. Of the 34 mesophilic isolates, 5 (15%), 16 (47%), and 3 (9%) displayed amylase, protease, and lipase activities, respectively. Among these isolates, the following species were identified based on their 16S rRNA gene sequences: Aneurinibacillus aneurinilyticus, Bacillus fortis, Bacillus subtilis, Brachybacterium paraconglomeratum, Brevibacterium otitidis, Dietzia maris, Pseudomonas xiamenensis, Staphylococcus lentus, Thermobifida fusca, Ureibacillus thermosphaericus, and Vagococcus lutrae. However, 15 isolates could not be identified as known taxa, thus indicating new bacterial taxa. Of these new taxa, it is likely that NoID A plays an important role in organic matter decomposition during composting based on its physiological characteristics. Sapporo sewage sludge compost contains a microbial ecosystem with novel bacterial biodiversity, comprising a high percentage of previously unrecognized species. This study improves our knowledge of the unique bacteria in sewage sludge compost, providing a future resource for bacterial genetic information and bacterial species of agricultural benefit.     

Biodegradation of kraft-lignin by <Emphasis Type="Italic">Bacillus</Emphasis> sp. isolated from sludge of pulp and paper mill

Eight bacterial strains were isolated on kraft lignin (KL) containing mineral salt medium (L-MSM) agar with glucose and peptone from the sludge of pulp and paper mill. Out of these, ITRC-S8 was selected for KL degradation, because of its fast growth at highest tested KL concentration and use of various lignin-related low molecular weight aromatic compounds (LMWACs) as sole source of carbon and energy. The bacterium was identified by biochemical tests as Gram-positive, rod-shaped and non-motile. Subsequent 16S rRNA gene sequencing showed 95% base sequence homology and it was identified as Bacillus sp. In batch experiments, a decrease in pH was observed initially followed by an increase till it reached an alkaline pH, which did not alter the culture growth significantly. The bacterium reduced the colour and KL content of 500 mg l⁻¹ KL in MSM, in the presence of glucose and peptone, at pH 7.6, temperature 30°C, agitation of 120 rpm and 6 days of incubation by 65 and 37% respectively. Significant reduction in colour and KL content in subsequent incubations is indicative of a co-metabolism mechanism, possibly due to initial utilization of added co-substrates for energy followed by utilization of KL as a co-metabolic. The degradation of KL by bacterium was confirmed by GC-MS analysis indicating formation of several LMWACs such as t-cinnamic acid, 3, 4, 5-trimethoxy benzaldehyde and ferulic acid as degradation products, which were not present in the control (uninoculated) sample. This favours the idea of biochemical modification of the KL polymer to a single monomer unit.     

Identification of bacteria contaminating pulp and a paper machine in a Canadian paper mill

Over 100 bacteria from pulp and slime samples in a Canadian paper mill were identified by partial sequencing of their 16S rDNAs. Seventy-one percent of the isolates could be assigned to a bacterial genus with a high level of confidence. Another 12% exhibited at least 95% similarity within their 16S rDNA sequence with unidentified organisms that originate from warm or wet environments. Pseudomonas, Bacillus, and Pseudoxanthomonas isolates were represented at a relatively high proportion in both pulp and slime samples. This is the first time that Pseudoxanthomonas strains have been isolated from pulp and slime samples on a paper machine. Electronic Publication     

Degradation of 2,4-dichlorophenol by Bacillus sp. isolated from an aeration pond in the Baikalsk pulp and paper mill (Russia)

A pure culture of 2,4-dichlorophenol (2,4-DCP)-degrading bacteria was isolated from a natural enrichment that had been adapted to chlorophenols in the aeration pond of the Baikalsk pulp and paper mill (Russia). The bacteria were identified by 16S rDNA intergenic region analysis, using PCR with universal primers. Comparative analysis of the 16S rDNA sequence (1545 bp) in the GenBank database revealed that these bacteria are related to Bacillus cereus GN1. Degradation of 2,4-DCP was studied using this culture in liquid medium under aerobic conditions, at initial concentrations of 20–560 μM 2,4-DCP. The 2,4-DCP degradation rates by B. cereus GN1 could be determined at concentrations up to 400 μM. However, higher concentrations of 2,4-DCP (560 μM) were inhibitory to cell growth.     

Bioremediation 2,4,6,-Trichlorophenol (2,4,6-TCP) by Shigella sp. S2 Isolated from Industrial Dumpsite

A bacterium that utilizes 2,4,6-trichlorophenol (2,4,6-TCP) as a sole source of carbon and energy was isolated from an industrial dumpsite, the bacterium designated as strain S2. Degradation was routinely monitored by observing growth analysis, chloride release assay, and ring cleavage activity and was further confirmed by gas chromatography (GC) analysis. The bacterium was found to degrade up to 90% of 2,4,6-TCP at 1.5 mM concentration. The bacteria were characterized morphologically, biochemically, and by 16S rRNA gene sequencing, which showed 99% sequence similarity with Shigella sp. This is the first report that Shigella sp. was able to degrade 2,4,6-TCP. This strain was found to be novel and a potential 2,4,6-TCP degrader. Further, this strain may be used for bioremediation of 2,4,6-TCP–containing waste in the environment.     

Morphological and Biochemical Properties of a Sphaerotilus sp. Isolated From Paper Mill Slimes

Four strains of filamentous bacteria were isolated from slimes collected in different paper mill factories. Morphological and physiological characterization of the isolates indicated an affiliation with the genus Sphaerotilus. However, while the physiological properties of the isolates were almost identical, pronounced physiological differences between the isolates and Sphaerotilus natans DSM 6575^T, DSM 565, and DSM 566 with respect to their ability to metabolize complex polysaccharides, sugars, polyalcohols, or organic acids as carbon sources were detected. In contrast to the analyzed culture collection strains of S. natans, all paper mill isolates were able to grow at elevated temperatures of up to 40°C. Comparative sequence analysis of nearly complete 16S ribosomal DNA (rDNA) sequences from the four new isolates demonstrated that the retrieved sequences were highly similar to each other (99.6 to 99.8% similarity) and to previously published partial 16S rDNA sequences of S. natans DSM 6575^T and ATCC 15291. Polyphasic characterization of the isolated Sphaerotilus strains revealed interesting adaptations of the strains to the environmental paper mill conditions with regard to temperature tolerance and utilization of cellulose and starch.     

Biodegradation and Decolorization of Pulp and Paper Mill Effluent by Anaerobic and Aerobic Microorganisms in a Sequential Bioreactor

Summary Paecilomyces sp. and Pseudomonas syringae pv myricae (CSA105) were isolated from sediment core of drainage of the pulp and paper mill industry. Fungi and bacteria were applied for treatment of pulp and paper mill effluent in a two-step and three-step fixed film sequential bioreactor containing sand and gravel at the bottom of the reactor for immobilization of microbial cells. Degradation of chlorinated phenols and formation of their metabolites were determined by high performance liquid chromatography. The microbes exhibited significant reduction in colour (88.5%), lignin (79.5%), chemical oxygen demand (87.2%) and phenol (87.7%) in two-step aerobic sequential bioreactor, and colour (87.7%), lignin (76.5%), chemical oxygen demand (83.9%) and phenol (87.2%) in three-step anaerobic-aerobic sequential bioreactor.     

Bioprospection and selection of bacteria isolated from environments contaminated with petrochemical residues for application in bioremediation

The use of microorganisms with hydrocarbon degrading capability and biosurfactant producers have emerged as an alternative for sustainable treatment of environmental passives. In this study 45 bacteria were isolated from samples contaminated with petrochemical residues, from which 21 were obtained from Landfarming soil contaminated with oily sludge, 11 were obtained from petrochemical industry effluents and 13 were originated directly from oily sludge. The metabolization capability of different carbon sources, growth capacity and tolerance, biosurfactant production and enzymes detection were determined. A preliminary selection carried out through the analysis of capability for degrading hydrocarbons showed that 22% of the isolates were able to degrade all carbon sources employed. On the other hand, in 36% of the isolates, the degradation of the oily sludge started within 18–48 h. Those isolates were considered as the most efficient ones. Twenty isolates, identified based on partial sequencing of the 16S rRNA gene, were pre-selected. These isolates showed ability for growing in a medium containing 1% of oily sludge as the sole carbon source, tolerance in a medium containing up to 30% of oily sludge, ability for biosurfactant production, and expression of enzymes involved in degradation of aliphatic and aromatic compounds. Five bacteria, identified as Stenotrophomonas acidaminiphila BB5, Bacillus megaterium BB6, Bacillus cibi, Pseudomonas aeruginosa, and Bacillus cereus BS20 were shown to be promising for use as inoculum in bioremediation processes (bioaugmentation) of areas contaminated with petrochemical residues since they can use oily sludge as the sole carbon source and produce biosurfactants.