J. Grant Burgess

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10 ^T and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10 ^T and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.

Problems with tin and copper antifouling compounds have highlighted the need to develop new environmentally friendly antifouling coatings. Bacteria isolated from living surfaces in the marine environment are a promising source of natural antifouling compounds. Four isolates were used to produce extracts that were formulated into ten water-based paints. All but one of the paints showed activity against a test panel of fouling bacteria. Five of the paints were further tested for their ability to inhibit the settlement of barnacle larvae, Balanus amphitrite , and algal spores of Ulva lactuca , and for their ability to inhibit the growth of U. lactuca . Two paints caused a significant decrease in the number of settled barnacles. One paint containing extract of Pseudomonas sp. strain NUDMB50-11, showed excellent activity in all assays. The antifouling chemicals responsible for the activity of the extract were isolated, using bioassay guided fractionation, and their chemical structures determined.

The bacterial consortium MPD‐M, isolated from sediment associated with Colombian mangrove roots, was effective in the treatment of hydrocarbons in water with salinities varying from 0 to 180 g L⁻¹. Where the salinity of the culture medium surpassed 20 g L⁻¹, its effectiveness increased when the cells were immobilized on polypropylene fibers. Over the range of salinity evaluated, the immobilized cells significantly enhanced the biodegradation rate of crude oil compared with free‐living cells, especially with increasing salinity in the culture medium. Contrary to that observed in free cell systems, the bacterial consortium MPD‐M was highly stable in immobilized systems and it was not greatly affected by increments in salinity. Biodegradation was evident even at the highest salinity evaluated (180 g L⁻¹), where biodegradation was between 4 and 7 times higher with immobilized cells compared to free cells. The biodegradation of pristane (PR) and phytane (PH) and of the aromatic fraction was also increased using cells immobilized on polypropylene fibers. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 145–153, 2002.

Bioactive agents produced by marine surface‐associated bacteria have been incorporated into experimental antifouling coatings. It was found that coating formulation can affect the action spectra of bacterial extracts involved. Little work has been done on the effect of permeability on the leaching of bioactive compounds from antifouling coatings. The formulations with 1.5% and 1.0% alginate produced significantly larger zones (p < 0.05) than with 0.5% alginate. Test formulations continued to leach active compounds after continuous immersion in seawater for 120 h. The bacterial strains used to produce the active compounds were identified by biochemical tests and/or the analysis of the DNA from a 500 bp section of the gene coding for the 16S rRNA subunit. Strains used were tentatively identified as Planococcus citreus, seven Bacillus strains (including three Bacillus pumilus and three Bacillus lautus strains), Cytophaga fucicola, Cytophaga uliginosa and a Pseudoalteromonas spp.

Ecological problems associated with current antifouling technologies have increased interest in the natural strategies that marine organisms use to keep their surfaces clean and free from fouling. Bacteria isolated from living surfaces in the marine environment have been shown to produce chemicals that are potential antifoulants. Active compounds from the cells and culture supernatant of two bacterial strains, FS‐55 and NudMB50–11, isolated from surface of the seaweed, Fucus serratus, and the nudibranch, Archidoris pseudoargus, respectively, were extracted using solid phase extraction. The extracts were combined with acrylic base paint resin and assayed for antifouling activity by measuring their ability to inhibit the growth of fouling bacteria. These formulations were found to be active against fouling bacteria isolated from marine surfaces. The formulation of antifouling paints that incorporate marine microbial natural products is reported here for the first time. This is a significant advance towards the production of an environmentally friendly antifouling paint that utilises a sustainable supply of natural biodegradable compounds.

Despite the ubiquity of bacteria in the marine environment little is known about the structure and function of microbial communities associated with marine ma‐croorganisms. Using a range of traditional and novel culture methods, 280 isolates of epiphytic marine bacteria from a range of marine algae were obtained in pure culture. Sixty of the 280 isolates (21%) exhibited antibiotic activity against a test battery of fouling bacteria. Fractionation of the culture supernatants derived from two strains, GB3 and AR55, indicated the presence of a multicomponent defence system consisting of both organic and water soluble components. In addition, eight out of 21 strains (38%) screened using a spectro‐photometric chemotaxis assay, produced metabolites capable of eliciting a negative chemotactic response in a motile fouling bacterium. A significant fraction of these isolates therefore have the potential to control the microbial population on the seaweed surface, either through inhibiting the growth, or influencing the tactic behaviour of potentially competing bacteria.