Genetic diversity of Paramecium species on the basis of multiple loci analysis and ITS secondary structure models.

Among ciliates, Paramecium has become a privileged model for the study of "species problem" particularly in the case of the "Paramecium aurelia complex" that has been intensely investigated. Despite extensive studies, the taxonomy of Paramecium is still challenging. The major problem is an uneven sampling of Paramecium with relatively few representatives of each species. To investigate species from the less discovered region (Pakistan), 10 isolates of Paramecium species including a standing-alone FT8 strain previously isolated by some of us were subjected to molecular characterization. Fragments of 18S recombinant DNA (rDNA), ITS1-5.8S-ITS2-5'LSU rDNA, cytochrome c oxidase subunit II, and hsp70 genes were used as molecular markers for phylogenetic analysis of particular isolates. The nucleotide sequences of polymerase chain reaction products of all markers were compared with the available sequences of relevant markers of other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all the nine strains had a very close relationship with Paramecium primaurelia except for the FT8 strain. FT8 consistently showed its unique position in comparison to all other species in the phylogenetic trees. Available sequences of internal transcribed spacer 1 (ITS1) and ITS2 and some other ciliate sequences from GenBank were used for the construction of secondary models. Two highly conserved helices supported by compensatory base changes among all ciliates of ITS2 secondary structures were found similar to other eukaryotes. Therefore, the most conserved 120 to 180 base pairs regions were identified for their comparative studies. We found that out of the three helices in ITS1 structure, helix B was more conserved in Paramecium species. Despite various substitutions in the primary sequence, it was observed that secondary structures of ITS1 and ITS2 could be helpful in interpreting the phylogenetic relationships both at species as well as at generic level.

Arsenic processing of yeast isolates IIB-As1 & IIB-As2 and production of glutathione under stress conditions.

Four arsenic resistant yeast were isolated from the industrial wastewater. Two strains IIB-As1 and IIB-As2 identified as Candida tropicalis and Saccharomyces cerevisiae, respectively. IIB-As1 and IIB-As2 showed maximum arsenic resistance. IIB-As1 showed maximum growth at 35 °C whereas it was 30 °C for IIB-As2. The yeast isolate showed typical growth curves, but arsenic extended the lag phase. Glutathione plays an important role in metal tolerance. In the present study, As increased the level glutathione and non-protein thiols in yeast isolates. Removal of As from supernatant was analyzed using the atomic absorption spectrophotometer. They removed arsenic from the medium after 72 h of incubation. Both yeast strains efficiently removed arsenic from the industrial effluent when used individually or in consortia.

Variability in secondary structure of 18S ribosomal RNA as topological marker for identification of Paramecium species.

Besides cytological and molecular applications, Paramecium is being used in water quality assessment and for determination of saprobic levels. An unambiguous identification of these unicellular eukaryotes is not only essential, but its ecological diversity must also be explored in the local environment. 18SrRNA genes of all the strains of Paramecium species isolated from waste water were amplified, cloned and sequenced. Phylogenetic comparison of the nucleotide sequences of these strains with 23 closely related Paramecium species from GenBank Database enabled identification of Paramecium multimicronucleatum and Paramecium jenningsi. Some isolates did not show significant close association with other Paramecium species, and because of their unique position in the phylogenetic tree, they were considered new to the field. In the present report, these isolates are being designated as Paramecium caudatum pakistanicus. In this article, secondary structure of 18SrRNA has also been analyzed as an additional and perhaps more reliable topological marker for species discrimination and for determining possible phylogenetic relationship between the ciliate species. On the basis of comparison of secondary structure of 18SrRNA of various isolated Paramacium strains, and among Paramecium caudatum pakistanicus, Tetrahymena thermophila, Drosophila melanogaster, and Homo sapiens, it can be deduced that variable regions are more helpful in differentiating the species at interspecific level rather than at intraspecific level. It was concluded that V3 was the least variable region in all the organisms, V2 and V7 were the longest expansion segments of D. melanogaster and there was continuous mutational bias towards G.C base pairing in H. sapiens.

Evaluation of consortia of microorganisms for efficient removal of hexavalent chromium from industrial wastewater.

The Chromium (Cr) uptake ability of Cr-resistant bacterium Bacillus thuringiensis, yeast Candida etschellsii, and a protozoan Stylonychia mytilus, isolated from industrial waste water, was evaluated individually and in different combinations. It was found that the three types of microorganisms grown together in a culture medium could collectively uptake 90% of Cr(6+) from the culture medium as against 82% by bacterium + protozoan or yeast + protozoan combined culture, each. Consortium of bacterium, yeast and ciliates therefore could make much more efficient inoculum for remediation of Cr-contaminated industrial waste water.

Heavy metal uptake by Euplotes mutabilis and its possible use in bioremediation of industrial wastewater.

A ciliate protozoan, Euplotes mutabilis, isolated from heavy metal laden industrial wastewater, has been shown to tolerate multiple heavy metals thus suggesting its significance in bioremediation of industrial effluents. This ciliate tolerated Zn(2+) up to 33 microg/mL, Cd(2+) up to 22 microg/mL and Ni(2+) up to 18 microg/mL. The ciliate could uptake 85% Zn(2+), 84% of Cd(2+) and 87% of Ni(2+) after 96 h of inoculation of growth medium containing 10 microg/mL of Zn(2+) and 5 microg/mL of Cd(2+) and Ni(2+), with actively growing ciliates. After 6 days of incubation the ciliate removed 87% Cd(2+), 92% Ni(2+), and 93% Zn(2+) from the wastewater. The heavy metal uptake capability of Euplotes mutabilis may be employed for metal detoxification operations.

Electrochemistry and microbiology of microbial fuel cells treating marine sediments polluted with heavy metals.

The industrial contamination of marine sediments with chromium, copper and nickel in Penang, Malaysia was addressed with bio-remediation, coupled with power generation, using in situ sediment microbial cells (SMFCs) under various conditions. The efficiency of aerated sediment microbial fuel cells (A-SMFCs) and non-aerated sediment microbial fuel cells (NA-SMFCs) was studied. The A-SMFCs generated a voltage of 580.5 mV between 50 and 60 days, while NA-SMFCs produced a voltage of 510 mV between 60 and 80 days. The cell design point for A-SMFCs was 2 kΩ, while for NA-SMFCs it was 200 Ω. In both SMFCs, the maximum current values relating to forward scanning, reverse scanning and oxidation/reduction peaks were recorded on the 80th day. The anode showed maximum additional capacitance on the 80th day (A-SMFC: 2.7 F cm-2; and NA-SMFC: 2.2 F cm-2). The whole cell electrochemical impedance using the Nyquist model was 21 Ω for A-SMFCs and 15 Ω for NA-SMFCs. After glucose enrichment, the impedance of A-SMFCs was 24.3 Ω and 14.6 Ω for NA-SMFCs. After 60 days, the A-SMFCs reduced the maximum amount of Cr(vi) to Cr(iii) ions (80.70%) and Cu(ii) to Cu(i) ions (72.72%), and showed maximum intracellular uptake of Ni(ii) ions (80.37%); the optimum remediation efficiency of NA-SMFCs was after 80 days toward Cr(vi) ions (67.36%), Cu(ii) ions (59.36%) and Ni(ii) ions (52.74%). Both SMFCs showed highest heavy metal reduction and power generation at a pH of 7.0. SEM images and 16S rRNA gene analysis showed a diverse bacterial community in both A-SMFCs and NA-SMFCs. The performance of A-SMFCs showed that they could be exercised as durable and efficient technology for power production and the detoxification of heavy metal sediments. The NA-SMFCs could also be employed where anaerobic fermentation is required.

Aetiology of shigellosis in northern Pakistan.

People of northern Pakistan face health hazards because of poor sanitation practices. Bacterial gastrointestinal infections are very common, and sometimes outbreaks occur. The present study was aimed at evaluating and analyzing infestation of Shigella spp. in patients with suspected gastroenteritis and ascertaining the status of antibiotic therapy. Five hundred and eighty-five faecal samples of patients with suspected gastroenteritis, referred to the District Headquarter Hospital Gilgit, were investigated for common enteropathogenic bacteria from July 1997 to September 1999. Seventy-seven (13.2%) of the faecal specimens were infected with different strains of Shigella spp., 61% of which were Shigella dysenteriae, 15.6% were S. flexneri, and 23.4% were Shigella sp. All Shigella strains were sensitive to ceftriaxone, cefotaxime, ciprofloxacin, and enoxacin. Sixty-one percent of the strains were resistant to both ampicillin and chloramphenicol, and 3.9% to ampicillin and nalidixic acid, while 10.4% were resistant to ampicillin alone and 14.3% to chloramphenicol only. Only 10.4% of the strains were sensitive to all the antibiotics tested. Sixty strains of Shigella spp. were processed for isolation of plasmids, and 58 (97%) of these antibiotic-resistant bacteria harboured at least one plasmid. The number of plasmids varied from 1 to 9. Escherichia coli C600 were transformed with the isolated plasmids. Transformants, containing 23-kb plasmid, resisted growth in media containing antibiotics, thereby indicating that antibiotic resistance is plasmid-borne. Based on the findings of the study, it is concluded that the infestation of Shigella spp. is high in northern Pakistan, the aetiological agents are highly resistant to chloramphenicol and ampicillin, and the antibiotic resistance is mediated by the 23-kb plasmid.

Isolation and characterization of arsenic resistant bacteria from wastewater.

The present study proposed the isolation of arsenic resistant bacteria from wastewater. Only three bacterial isolates (MNZ1, MNZ4 and MNZ6) were able to grow in high concentrations of arsenic. The minimum inhibitory concentrations of arsenic against MNZ1, MNZ4 and MNZ6 were 300 mg/L, 300 mg/L and 370 mg/L respectively. The isolated strains showed maximum growth at 37 °C and at 7.0 pH in control but in arsenite stress Luria Bertani broth the bacterial growth is lower than control. All strains were arsenite oxidizing. All strains were biochemically characterized and ribotyping (16S rRNA) was done for the purpose of identification which confirmed that MNZ1 was homologous to Enterobacter sp. while MNZ4 and MNZ6 showed their maximum homology with Klebsiella pneumoniae. The protein profiling of these strains showed in arsenic stressed and non stressed conditions, so no bands of induced proteins appeared in stressed conditions. The bacterial isolates can be exploited for bioremediation of arsenic containing wastes, since they seem to have the potential to oxidize the arsenite (more toxic) into arsenate (less toxic) form.

Isolation and characterization of arsenic resistant bacteria from wastewater

The present study proposed the isolation of arsenic resistant bacteria from wastewater. Only three bacterial isolates (MNZ1, MNZ4 and MNZ6) were able to grow in high concentrations of arsenic. The minimum inhibitory concentrations of arsenic against MNZ1, MNZ4 and MNZ6 were 300 mg/L, 300 mg/L and 370 mg/L respectively. The isolated strains showed maximum growth at 37 ºC and at 7.0 pH in control but in arsenite stress Luria Bertani broth the bacterial growth is lower than control. All strains were arsenite oxidizing. All strains were biochemically characterized and ribotyping (16S rRNA) was done for the purpose of identification which confirmed that MNZ1 was homologous to Enterobacter sp. while MNZ4 and MNZ6 showed their maximum homology with Klebsiella pneumoniae. The protein profiling of these strains showed in arsenic stressed and non stressed conditions, so no bands of induced proteins appeared in stressed conditions. The bacterial isolates can be exploited for bioremediation of arsenic containing wastes, since they seem to have the potential to oxidize the arsenite (more toxic) into arsenate (less toxic) form.