Kazuo Kakii

Coaggregation among Nonflocculating Bacteria Isolated from Activated Sludge

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

Coaggregation between Acinetobacter johnsonii S35 and Microbacterium esteraromaticum strains isolated from sewage activated sludge

The extent and nature of intergeneric coaggregations among non-flocculating sludge bacteria were studied through examination of the coaggregation abilities of Acinetobacter johnsonii S35 with two other strains of non-flocculating sludge bacteria (Microbacterium esteraromaticum S38 and M. esteraromaticum S51). At first, the effect of electrolyte concentration as well as the addition of EDTA and proteases on coaggregation were studied. Changes in electrolyte concentration had little effect on the aggregation index (A.I.). However, the A.I. was reduced by 35-45% (addition of 2 mM EDTA) and 15-58% (addition of 0.2 mg/ml Actinase E) within 1 h depending upon the bacterial pair involved. Furthermore, the effect of pretreating either A. johnsonii S35 or its partners with Actinase E and periodate, respectively, was studied. The results indicate that these coaggregations involve a protein on the partners surface that may interact with carbohydrate or a carbohydrate-containing moiety on the surface of A. johnsonii S35.

Intergeneric coaggregations among Oligotropha carboxidovorans and Acinetobacter species present in activated sludge

The coaggregation traits of two pairs of sewage sludge bacteria were tested and characterized. Oligotropha carboxidovorans S23 coaggregated with two strains of the genus Acinetobacter viz. Acinetobacter junii S33 (56%) and Acinetobacter johnsonii S35 (99%). Coaggregates of O. carboxidovorans S23 and A. junii S33 were small (20-40 microm), weak and susceptible to EDTA and a commercial protease (Actinase E). Actinase/periodate pretreatment of the partners prior to coaggregation revealed that interaction in this case was mediated by protein surface components. Coaggregates of O. carboxidovorans S23 and A. johnsonii S35 were large (above 100 microm), strong and not deflocculated by EDTA or Actinase E. Only periodate pretreatment of A. johnsonii S35 prevented this coaggregation indicating a role for a carbohydrate-containing moiety without the involvement of protein components. The potential mechanisms and strength of bacterial coaggregations seem to be pair dependent.

Isolation and Growth Characteristics of Nitrilotriacetate-Degrading Bacteria

Abstract Two bacterial strains that degrade nitrilotriacetate (NTA) were isolated from NTA-acclimatized activated sludge. These bacteria grew well in NTA medium with optimal pH around 7. The growth rate constants of the bacteria, strains N-2 and N-5, were 0.046 h−1 and 0.11 h−1 at the concentration of 0.1% NTA (pH 7.0, 25°C), respectively. The growth of each bacterium was inhibited at high concentrations NTA. The growth rate decreased roughly linearly with increasing concentration of NTA. The strains N-2 and N-5 showed maximal cell growth at the concentrations of 0.2% and 0.25% NTA, respectively. The strain N-2 would not grow at the concentration of 0.5% NTA. On the other hand, the strain N-5 showed a little growth under the same conditions. Also, the bacterial growth was almost completely inhibited when divalent metal ions such as Mg++, Ca++, and Fe++ were omitted from the culture medium, or slightly excess EDTA (1 mM) was added to the medium. These results suggest that the bacterial growth inhibition at high concentration of NTA is caused by the sequestration of metal ions in the medium.

Intergeneric coaggregation of non-flocculating Acinetobacter spp. isolates with other sludge-constituting bacteria

The ability of non-flocculating Acinetobacter spp. isolates (A. johnsonii S35 and A. junii S33) to coaggregate with other sludge-constituting bacteria obtained from a sewage sludge sample was assessed by a spectrophotometric assay. A. johnsonii S35 showed significant coaggregation (aggregation index (AI)=70 to 99%) with 7 strains, Bacillus cereus, Blastomonas natatoria, Mycobacterium sp., Thermomonas brevis, and Enterobacteriaceae family, identified by sequencing of 16S rRNA genes. A. junii S33 coaggregated with 4 strains, which were also coaggregating partners of A. johnsonii S35, as indicated by AI values of 59-93%. The microbial adhesion to hydrocarbon (MATH) assay showed that both A. johnsonii S35 and A. junii S33 possess a hydrophobic character as indicated by their relative cell surface hydrophobicity (RCSH), 88 and 61%, respectively. A good correlation was observed between the degree of coaggregation of the Acinetobacter spp. isolates and the RCSH of their partner strains. The cells of an A. johnsonii S35 spontaneous mutant and A. johnsonii IAM1517, which had a hydrophilic cell surface, did not coaggregate with the other examined isolates. These results demonstrated that hydrophobic interaction plays an important role in the intergeneric coaggregation of Acinetobacter spp. isolates with other sludge-constituting bacteria.

Involvement of Ca2+ in the flocculation of Kluyvera cryocrescens KA-103

Abstract Kluyvera cryocrescens KA-103 showed a dispersed growth in Ca 2+ -free Polypepton medium, but formed flocs on addition of a sufficient concentration of Ca 2+ to the bacterial cell suspension. Therefore, calcium adsorption properties and flocculation conditions were investigated using bacterial cells cultured in the Ca 2+ -free Polypepton medium. The bacterium required 1.5 mM Ca 2+ or more for good flocculation (F>90%), but a cooperative effect of Na + and Ca 2+ on good flocculation was observed at lower concentrations of Ca 2+ . The Langmuir adsorption isotherm was used to describe the adsorption of Ca 2+ by the bacterial cells.

Extracellular polymers in relation to settling properties of activated sludge

Abstract The effects of cetyltrimethylammonium bromide (CTAB) and acid on the settling properties of activated sludge samples from various sources were investigated. The addition of CTAB or acid to mixed liquor resulted in an increase of zone settling velocity (ZSV) and a reduction of 30-min settled sludge volume (SV 30 ). The higher the amount of extracellular polymers (ECPs) extracted from activated sludge, the higher the effectiveness. The amount of cetyltrimethylammonium ion (CTA + ) adsorbed by activated sludge was decreased by the extraction of ECPs and the extracted ECPs precipitated on addition of CTAB. These results indicated that the adsorption sites for CTA + are acidic functional groups in the ECPs. The Langmuir adsorption isotherm was employed successfully to describe the adsorption of CTA + by activated sludge. The equilibrium constants ( K ) for complexes formed between CTA + and the adsorption sites of activated sludge ranged from 10 5.1 to 10 6.0 .

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

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

Camparison of mucilage polysaccharides extracted from sewage activated sludge

Abstract The sugar composition of mucilage polysaccharides extracted from activated sludge from five different sewage treatment plats were compared. All the polysaccharides contained rhamnose, fucose, arabisone, xylose, mannose, galactose, glucose, amino sugars, and uronic acids in similar proportions, especially in the neutral sugar fraction. The main components were rhamnose (12–18%), mannose (14–21%), galactose (16–19%), and glucose (15–23%). No significant changes was observed in the sugar composition of activated sludge from a sewage treatment plant over a period of more than one year. Recovery of the mucilage polysaccharides fell to 46% of the initial amount when activated sludge was digested aerobically for 10 days, but the sugar composition was not affected.

Toxic effects of Hg2+ to a Hg2+-resistant Pseudomonas in the presence of halide ions

Abstract A Hg 2+ -resistant Pseudomonas which can utilize glycine as a sole carbon and nitrogen source was isolated from a municipal sewage treatment plant, and the toxicity of Hg 2+ in the presence of halide ions was examined. In glycine medium without halide ions, bacterial growth was scarcely influenced even at an initial Hg 2+ concentration of 25 μM. The viable cell count decreased when adsorbed Hg 2+ was more than 300 μmol·g-cell −1 . The toxicity of Hg 2+ was enhanced when the halide ion concentration was such that the [HgX 2 ] 0 complex ( X =Br − or I − ) in the solution became dominant. In the presence of [HgBr 2 ] 0 complex, complete growth inhibition of unacclimatized cells was observed at 2–3 μM Hg 2+ . A sudden decrease in the viable cell count was evident when adsorbed Hg 2+ was more than 15 μmol·g-cell −1 . Growth of the isolate pre-incubated in glycine medium containing 25 μM Hg 2+ (acclimatized cells) was also inhibited completely at 10 μM Hg 2+ in the presence of Br − . The adsorption and toxicity of Hg 2+ decreased with increasing halide ion concentration in which [HgBr 3 ] − or [HgBr 4 ] 2− complexes exist dominantly. These results indicate that toxicity of Hg 2+ was enhanced by the formation of [HgX 2 ] 0 complex and was not governed by the amount of Hg 2+ adsorbed to the cells.