Chlorination-induced cellular damage and recovery in marine microalga, Chlorella salina.

Power plants employ chlorination for controlling biofouling in the cooling water system. Phytoplankton drawn into the cooling water system could be impacted by chemical stress induced by the oxidizing biocide. It is likely that microalgae, being sensitive to chlorine, could suffer damage to their cellular structure and function. In this study, we present data on the effect of in-use concentrations of chlorine on the unicellular microalga, Chlorella salina. Chlorophyll autofluorescence was measured in terms of mean fluorescence intensity per cell for rapid assessment of toxicity. Viability of the cells exposed to chlorine was determined by fluorescein diacetate staining. Functionality of the photosynthetic machinery was assessed by gross primary productivity. Results from the study, which combined confocal laser scanning microscopy with image analysis, showed a significant dose-dependant reduction in chlorophyll autofluorescence, esterase activity and gross primary productivity in chlorine-treated cells. Interestingly, the cells injured by chlorination could not recover in terms of autofluorescence, esterase activity or productivity even after 18 h incubation in healthy media. Among the test points evaluated, esterase activity appeared to be sensitive for determining the chlorination-induced impact. Our results demonstrate that low-dose chlorination causes significant decrease in chlorophyll autofluorescence, intracellular esterase activity and primary productivity in Chlorella cells.

P-nitrophenol biodegradation by aerobic microbial granules.

Mixed microbial consortia in the form of aerobic microbial granules (AMG) capable of xenobiotic degradation can be developed from activated sludge or by adaptation of microbial granules pre-grown on labile carbon sources. Both of these approaches were investigated for the cultivation of AMG capable of p-nitrophenol (PNP) biodegradation. Attempts to cultivate AMG from activated sludge using PNP as the sole carbon source were not successful due to poor microbial growth and washout of the inoculated activated sludge. As part of the second approach, parallel sequencing batch reactors (SBRs) were inoculated with pre-grown AMG and operated by feeding both acetate and PNP together (RA), PNP alone (RB) or acetate alone (RC). Acetate/PNP mineralization and nitrification were monitored in the three SBRs. PNP biodegradation was quickly established in both RA and RB. PNP removal rates were found to be 47 and 55 mg g VSS(-1) h(-1) in RA and RB, respectively. PNP biodegradation during the SBR cycle consisted of distinct lag, exponential and deceleration phases. However, with higher concentrations of PNP (>50 mg l(-1)), disintegration of granules was observed in RA and RB. When PNP was the sole carbon source, it inhibited the development of aerobic granules from activated sludge and caused disintegration of pre-cultivated aerobic granules. When PNP was the co-substrate along with acetate, the structural and functional integrity (including nitrification) of the granular sludge was maintained. This report highlights the importance of a labile co-substrate for maintaining the physical and functional integrity of granular sludge, when used for toxic waste degradation.

Denitrification of synthetic concentrated nitrate wastes by aerobic granular sludge under anoxic conditions.

The aim of the present work was to determine the denitrification potential of aerobic granular sludge for concentrated nitrate wastes. We cultivated mixed microbial granules in a sequencing batch reactor operated at a superficial air velocity of 0.8 cm s(-1). The denitrification experiments were performed under anoxic conditions using serum bottles containing synthetic media with 225-2250 mg L(-1) NO3-N. Time required for complete denitrification varied with the initial nitrate concentration and acetate to nitrate-N mass ratio. Complete denitrification of 2250 mg L(-1) NO3-N under anoxic conditions was accomplished in 120 h. Nitrite accumulation was not significant (<5 mg N L(-1)) at initial NO3-N concentrations below 677 mg L(-1). However, denitrification of higher concentrations of nitrate (≥900 mg N L(-1)) resulted in buildup of nitrite. Nevertheless, nitrite buildups observed in present study were relatively lower compared to that reported in previous studies using flocculent activated sludge. The experimental results suggest that acetate-fed aerobic granular sludge can be quickly adapted to treat high strength nitrate waste and can thus be used as seed biomass for developing high-rate bioreactors for efficient treatment of concentrated nitrate-bearing wastes.

Alkyl-methylimidazolium ionic liquids affect the growth and fermentative metabolism of Clostridium sp.

In this study, the effect of ionic liquids, 1-ethyl-3-methylimidazolium acetate [EMIM][Ac], 1-ethyl-3-methylimidazolium diethylphosphate [EMIM][DEP], and 1-methyl-3-methylimidazolium dimethylphosphate [MMIM][DMP] on the growth and glucose fermentation of Clostridium sp. was investigated. Among the three ionic liquids tested, [MMIM][DMP] was found to be least toxic. Growth of Clostridium sp. was not inhibited up to 2.5, 4 and 4 g L(-1) of [EMIM][Ac], [EMIM][DEP] and [MMIM][DMP], respectively. [EMIM][Ac] at <2.5 g L(-1), showed hormetic effect and stimulated the growth and fermentation by modulating medium pH. Total organic acid production increased in the presence of 2.5 and 2 g L(-1) of [EMIM][Ac] and [MMIM][DMP]. Ionic liquids had no significant influence on alcohol production at <2.5 g L(-1). Total gas production was affected by ILs at ≥ 2.5 g L(-1) and varied with type of methylimidazolium IL. Overall, the results show that the growth and fermentative metabolism of Clostridium sp. is not impacted by ILs at concentrations below 2.5 g L(-1).

Rhamnolipid mediated disruption of marine Bacillus pumilus biofilms.

Removal of detrimental biofilms from surfaces exposed in the marine environment remains a challenge. A strain of Bacillus pumilus was isolated from the surface of titanium coupons immersed in seawater in the vicinity of Madras Atomic Power Station (MAPS) on the East coast of India. The bacterium formed extensive biofilms when compared to species such as Bacillus licheniformis, Pseudomonas aeruginosa PAO1 and Pseudomonas aureofaciens. A commercially available rhamnolipid was assessed for its ability to inhibit adhesion and disrupt pre-formed B. pumilus biofilms. The planktonic growth of B. pumilus cells was inhibited by concentrations >1.6mM. We studied the effect of various concentrations (0.05-100mM) of the rhamnolipid on adhesion of B. pumilus cells to polystyrene microtitre plates, wherein the effectiveness varied from 46 to 99%. Biofilms of B. pumilus were dislodged efficiently at sub-MIC concentrations, suggesting the role of surfactant activity in removing pre-formed biofilms. Scanning electron microscopy (SEM) confirmed the removal of biofilm-matrix components and disruption of biofilms by treatment with the rhamnolipid. The results suggest the possible use of rhamnolipids as efficient anti-adhesive and biofilm-disrupting agents with potential applications in controlling biofilms on surfaces.

Biofilm formation by Pseudoalteromonas ruthenica and its removal by chlorine.

The distribution of a recently described marine bacterium, SBT 033 GenBank Accession No. AY723742), Pseudoalteromonas ruthenica, at the seawater intake point, outfall and mixing point of an atomic power plant is described, and its ability to form biofilm was investigated. The effectiveness of the antifouling biocide chlorine in the inactivation of planktonic as well as biofilm cells of P. ruthenica was studied in the laboratory. The results show that the planktonic cells were more readily inactivated than the cells enclosed in a biofilm matrix. Viable counting showed that P. ruthenica cells in biofilms were up to 10 times more resistant to chlorine than those in liquid suspension. Using confocal laser scanning microscopy it was shown that significant detachment of P. ruthenica biofilm developed on a glass substratum could be accomplished by treatment with a dose of 1 mg l-1 chlorine. Chlorine-induced detachment led to a significant reduction in biofilm thickness (up to 69%) and substratum coverage (up to 61%), after 5-min contact time. The results show that P. ruthenica has a remarkable ability to form biofilms but chlorine, a common biocide, can be used to effectively kill and detach these biofilms.

Dual labeling of Pseudomonas putida with fluorescent proteins for in situ monitoring of conjugal transfer of the TOL plasmid.

We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor P. putida cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies.