P. Sriyutha Murthy

16S rDNA sequence analysis of culturable marine biofilm forming bacteria from a ship's hull

Marine bacteria from the hull of a ship in the form of biofilms or microfouling were isolated, cultured, and identified by phylogenetic analysis using 16S rDNA sequences. With an average length of 946 bp, all the 16 sequences were classified using the Ribosomal database project (RDP) and were submitted to the National Center for Biotechnology Information. Phylogenetic analysis using 16S rDNA sequences indicated that the 16 strains belonged to the Firmicutes (IK-MB6 Exiguobacterium aurantiacum, IK-MB7 Exiguobacterium arabatum, IK-MB8 Exiguobacterium arabatum, IK-MB9 Jeotgalibacillus alimentarius, IK-MB10 Bacillus megaterium, IK-MB11 Bacillus pumilus, IK-MB12 Bacillus pumilus, IK-MB13 Bacillus pumilus, IK-MB14 Bacillus megaterium), High GC, Gram-positive bacteria (IK-MB2 Micrococcus luteus, IK-MB5 Micrococcus luteus, IK-MB16 Arthrobacter mysorens), G-Proteobacteria (IK-MB3 Halomonas aquamarina, IK-MB15 Halotalea alkalilenta), CFB group bacteria (IK-MB1 Myroides odoratimimus), and Enterobacteria (IK-MB4 Proteus mirabilis). Among the 16 strains, representatives of the Firmicutes were dominant (56.25%) compared to the high GC, Gram-positive bacteria (18.75%), G-Proteobacteria (12.5%), CFB group bacteria (6.25%), and Enterobacteria (6.25%). Analysis revealed that majority of marine species found in marine biofilm are of anthropogenic origin.

Effect of Biofouling on Stability of Polycarbonate in Tropical Seawater

The effect of biofouling on physical, chemical and mechanical changes of Bisphenol A polycarbonate im- mersed at a depth of three meters at two different locations (Port and FSI, Chennai, India) in the Bay of Bengal Sea over a period of six months was the subject of this study. Biofouling in terms of total suspended solids, organic matter and total viable count was higher at Port than at FSI, probably because of higher dissolved oxygen at the former than at the latter site. An increase in glass transition temperature (from 133°C to 147 and 144°C at Port and FSI respectively) was observed in the samples indicating loss of amorphous region and conformational change in the polymer. A two fold decrease in ten- sile strength, a 33% decrease in contact angle and, a reduction in the tertiary methyl and carbonate carbonyl indices were observed. These findings indicate that polycarbonate undergoes a combination of biodeterioration and biodegradation un- der these conditions.

Gallium oxide nanoparticle induced inhibition of bacterial adhesion and biofilm formation

Toxicity of nano scaled Ga2O3 and functionalized (carboxyl -COOH; amine -NH) nanoparticles was examined against a gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria. NP concentrations in the range of 0.25 to 25 µg/mL were tested. Strain specificity in antimicrobial activity was observed. Ga2O3 treatments resulted in 26% inhibition of growth of planktonic cells in S. aureus at the lowest concentration of 0.25 µg/mL. Carboxyl functionalization resulted in a higher percentage of inhibition than that for amine functionalization in the growth of planktonic cells. In comparison, the gram negative strain (E. coli) has shown an increase in planktonic cell growth on treatment with Ga2O3 nanoparticles. The increase is lower in case of Ga2O3 treatment alone than those observed for COOH and NH modification. In contrast to planktonic cells, Ga2O3 and its functional groups retard biofilm formation even when high percentage of planktonic S. aureus and E. coli cells have survived the treatment. Higher percentage inhibition of biofilms is observed with both the functional groups. In comparison, higher percentage inhibition of biofilms is observed in the case of E. coli at the lowest concentration tested, when more than 100% of the planktonic cells survived the treatment. Results indicate that Ga2O3 may be involved in interfering with the molecular mechanisms.

Antibiofilm activity of nano sized CuO

Antibiofilm properties of bulk and nano-sized copper oxide (CuO) were investigated using four species of bacteria viz: Staphylococcus aureus, E. coli, Pseudomonas aeuriginosa (PAO1) and Pseudomonas putida. The effect of both commercial and synthesized nanoparticles on biofilm formation was investigated using different concentrations 0.02, 0.12, 0.24, 0.5, 1.0, 1.2, 1.4 & 1.8 µg / mL). Both bulk (91% reduction) and nano-sized (93% reduction) particles significantly reduced biofilm formation in all the four species of bacteria. Concentration and size/shape of nanoparticles seemed to influence biofilm inhibition. Increase in NP concentration from 0.02 to 1.8 µg/mL resulted in a marginal increase in inhibition. While a similar increase in concentration of bulk particles, resulted in 18% decrease in biofilm inhibition. Differences in biofilm inhibition between bulk and nano-sized particles were marginal at low concentrations (0.02, 0.12 & 0.24 µg/mL), whereas prominent differences were observed from concentration of 0.5 µg/mL onwards for the bulk particles. The decrease in inhibition efficiency of bulk nanoparticles with increase in concentration may be attributed to the differences in size, shape, aggregation and settling behaviour. In general, higher efficiency was observed with S. aureus, E. coli and P. putida biofilms, whereas P. aeuriginosa cells showed more resistance in the presence of CuO nanoparticles. Results of the present study indicated that size and shape of the nanoparticles, seem to have an influence on the effective concentration, required for inhibition of biofilms.

Chlorine dioxide as an alternative antifouling biocide for cooling water systems: Toxicity to larval barnacle Amphibalanus reticulatus (Utinomi)

Chlorine dioxide (ClO2) is seen as an effective alternative to chlorine, which is widely used as an antifouling biocide. However, data on its efficacy against marine macrofoulants is scanty. In this study, acute toxicity of ClO2 to larval forms of the fouling barnacle Amphibalanus reticulatus was investigated. ClO2 treatment at 0.1mg/L for 20min elicited 45-63% reduction in naupliar metamorphosis, 70% inhibition of cyprid settlement and 80% inhibition of metamorphosis to juveniles. Increase in concentration to 0.2mg/L did not result in any significant difference in the settlement inhibition or metamorphosis. Treatment with 0.2mg/L of ClO2 elicited substantial reduction in the settlement of barnacle larvae compared to control. The study indicates the possibility of using ClO2 as an alternative antifouling biocide in power plant cooling water systems. However, more work needs to be done on the environmental effects of such switchover, which we are currently undertaking.

Chlorination induced damage and recovery in marine diatoms: Assay by SYTOX® Green staining

Phytoplankton entrained into cooling water systems of coastal power stations are subjected to acute chemical stress due to biocides (chlorine) used for biofouling control. They are subsequently released into the environment, where they may survive/recover or succumb. Experiments were conducted to evaluate the susceptibility of a centric (Chaetoceros lorenzianus) and pennate (Navicula sp.) diatom to in-plant administered concentrations of chlorine (0.2-0.5mg/L, TRO). Viability of cells exposed to chlorine was assessed by SYTOX® Green fluorimetry and was compared with other conventional end points like total cell counts, chlorophyll a content and cellular autofluorescence. Results showed a concentration-dependant reduction in viability, chlorophyll a and autofluorescence. C. lorenzianus cells were more susceptible to chlorine compared to Navicula sp. SYTOX® Green staining appears to be a sensitive method to assess chlorine-induced damages. The data show that in-use levels of chlorination can potentially impact entrained organisms; however, they can recover when returned to coastal waters.

Polydimethyl siloxane based nanocomposites with antibiofilm properties for biomedical applications.

Polydimethyl siloxane (PDMS) is an excellent implant material for biomedical applications, but often fails as it is prone to microbial colonization which forms biofilms. In the present study CuO, CTAB capped CuO, and ZnO nanoparticles were tested as nanofillers to enhance the antibiofilm property of PDMS against Staphylococcus aureus and Escherichia coli. In general S. aurues (Gram positive and more hydrophobic) favor PDMS surface than glass while E. coli (Gram negative and more hydrophilic) behaves in a reverse way. Incorporation of nanofillers renders the PDMS surface antibacterial and reduces the attachment of both bacteria. These surfaces are also not cytotoxic nor show any cell damage. Contact angle of the material and the cell surface hydrophobicity influenced the extent of bacterial attachment. Cell viability in biofilms was dependent on the antimicrobial property of the nanoparticles incorporated in the PDMS matrix. Simple regression relationships were able to predict the bacterial attachment and number of dead cells on these nanocomposites. Among the nanocomposites tested, PDMS incorporated with CTAB (cetyl trimethylammonium bromide)-capped CuO appears to be the best antibacterial material with good cyto-compatibility.