Novel coaggregating microbial consortium: testing strength for field applications.

Acinetobacter johnsonii S35 has an ability to coaggregate with Oligotropha carboxidovorans S23 when grown with favorable carbon substrate and nutrient-rich growth media. However, nutrient-deficient situations or poorly accessible carbon substrates in waste streams could trigger changes in the cell surface properties and thus modify the coaggregation behavior. Thus, in the present study, we test the stability of these novel interactions under various stresses that might transpire in the waste streams. The effect of mineral-component deficiency and shortage of substrate on the growth, cell surface hydrophobicity and coaggregation ability of S35 and O. carboxidovorans S23 has been investigated. As compared to the control, growth of S35 was adversely affected in mineral-deficient medium; the cells had lower hydrophobicity but displayed only slightly lower aggregation index (AI, 75-84%) as compared to control (92%). S35 grown on marginally supplemented Sterile Filtrate of Activated Sludge (SFAS) could also grow well and form larger stable coaggregates with S23. S35 grown with mineral-deficient medium and with the supplemented SFAS still posses the ability to coaggregate with S23. Hence the coaggregation ability of S35 strain is constitutive and is not significantly affected by environmental factors. It would be desirable to work out suitable formulations of such robust and environmentally relevant microbes.

Effect of surfactants on stability of Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 coaggregates.

The effect of anionic (sodium dodecyl sulphate or SDS) and cationic (cetyltrimethylammonium bromide or CTAB) surfactants on the stability of binary bacterial coaggregates comprising Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 (both sewage sludge isolates) was studied and compared with that on the complex sewage sludge flocs. Both SDS and CTAB enhanced the bacterial coaggregation at their lower concentrations of 0.2 and 0.07 mg ml(-1), respectively. However, complete deflocculation of coaggregates was observed at 1 mg ml(-1) SDS and 0.3 mg l(-1) CTAB concentrations. Further, sewage sludge flocs did not deflocculate in the presence of CTAB, although a concentration-dependent deflocculation was observed in the presence of SDS. A. johnsonii S35 and O. carboxidovorans S23 cells were separately pretreated (prior to coaggregation) with the surfactants. In spite of the partial (complete) loss of viability during SDS (CTAB) pretreatment, washed cells still retained hydrophobic character and displayed significant coaggregation (aggregation index ranging from 84% to 97% in comparison to 96% in the case of non-treated cells), demonstrating reversibility of the surfactant induced deflocculation. Further, when exposed to lower concentration of surfactants (0.2 mg ml(-1) SDS), coaggregates were more resistant (76% viability) as compared to the individual partner (S35: 52%; S23: 39% viability). Since the coaggregates are stable and provide protection from surfactants at lower concentrations (those normally expected in the sewage treatment plants), their presence as well as a sustained role in the sewage sludge bioflocculation is evident.

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.

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 partner's surface that may interact with carbohydrate or a carbohydrate-containing moiety on the surface of A. johnsonii S35.

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.

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.

Pair-dependent co-aggregation behavior of non-flocculating sludge bacteria.

Two strains of non-flocculating sewage sludge bacteria (Xanthomonas sp. S53 and Microbacterium esteraromaticum S51) showed 91% and 77% co-aggregation, respectively, with Acinetobacter johnsonii S35 using a spectrophometric assay. The co-aggregates in case of Xanthomonas sp. S53 and A. johnsonii S35 were above 100 microm and stable against EDTA (2 mM) and a commercial protease (0.2 mg ml(-1)). Protease/periodate pretreatment of the partners did not affect this co-aggregation. On the other hand, co-aggregates of M. esteraromaticum S51 and A. johnsonii S35 (50-70 microm) were deflocculated by EDTA or protease. Protease pretreatment of M. esteraromaticum S51 and periodate pretreatment of A. johnsonii S35 prevented their co-aggregation with respective untreated partners. The potential co-aggregation mechanisms of A. johnsonii S35 varied depending upon the other partner involved.

Stability of bacterial coaggregates in extreme environments.

The stability of coaggregates formed by the interaction of Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23, both of which are nonflocculating sludge bacteria, was evaluated by a spectrophotometric assay of aggregation index and phase contrast microscopy. Exposure of coaggregates to conditions of various pH (3-11) or temperature (10-5 degrees C) did not cause significant deflocculation and high aggregation index (85-93%) was still maintained as compared to 93.3% under normal conditions. Further, heat pretreatment of A. johnsonii S35 and O. carboxidovorans S23 cells at 60 or 80 degrees C did not prevent their subsequent coaggregation indicating that the interacting cell surface structures are heat-resistant. However, dual exposure of coaggregates to low temperature (10 degrees C) and extreme pH caused disruption of coaggregates coupled with the reduction in aggregation index below 28% (pH 3) and 49% (pH 11). Use of sterile filtrate of settled sewage (SFSS) in place of electrolyte solution as coaggregating medium produced usual large coaggregates (above 100 microm) indicating reproducibility of this coaggregation in sewage treatment plants. SFSS coaggregates incurred lesser deflocculation (50 and 85%, respectively) at extreme pH (3 and 11, respectively) and low temperature. This study demonstrated that although bacterial coaggregates are strong and stable against individual pH or temperature stress, a dual stress might deteriorate the performance and quality of treated waters.

Coaggregation among nonflocculating bacteria isolated from activated sludge.

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.