Biofilm development was monitored over a period of 44 days in tanks receiving ozonated and non-ozonated water using microscopy and molecular based techniques. Bacterial groups detected in the biofilm were: Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Planctomycetales and the Cytophaga/Flavobacterium branch of phylum Bacteroidetes.
Denaturing gradient gel electrophoresis (DGGE) sequencing and fluorescence in situ hybridization (FISH) were used to identify bacteria.
For each treatment, eight-well microscope slides (for FISH) and regular microscope slides (for DGGE) microscope glass slides were placed in small-scale aquaculture tanks supplied with filtered, ozonated or non-ozonated seawater at a constant water flow-through of 1.0 to 1.2 Lmin-1, to which Artemia were added. Thereafter, Artemia were exchanged every day by removal of residual animals and addition of freshly harvested ones; at the same time dead Artemia and particulate matter was siphoned off the bottom of each tank. Mortality rates of Artemia were monitored throughout the experiment. Eight-well microscope slides were collected 1 h (day 0) and 24 h (day 1) after the first addition of Artemia to the tanks. Subsequently, sampling was continued every second day (days 3, 5, 7, 9, 11, 14, 16, 18 and 20) and then weekly (days 28, 36 and 44).
Probes used (and specificity): EUB338b (most bacteria); EUB338-IIb (additional bacteria - Planctomycetales); EUB338-IIIb IIb (additional bacteria - Verrucomicrobiales); NON338 (Antisense of EUB338); ALF1B (Alphaproteobacteria and some other bacteria ); BET42ad (Betaproteobacteria); GAM42ad (Gammaproteobacteria); CF319a (Cytophaga/Flavobacteria branch of Bacteroidetes); GV (Vibrio); PLA886d,e and PLA46e (Planctomycetales); G123d (Thiothrix).
Phylogenetic affiliation of sequences retrieved from bands excised from DGGE profiles of biofilm:
Sequence name (accession no.) environment : Clone b1uc29 (EF206937) bleached coral; Clone A714001 (AY907796) Arabian Sea picoplankton; Clone JTB23 (AB015248) deep cold-seep sediment; Rhodovulum sulfidophilum ATCC 35886 (DQ342323) marine mud flats; Clone b2ub58 (EF206984) bleached coral; Roseobacter sp. 49Xb1 (EU090129) stomach of Nyctiphanes simplex; Clone BBD (DQ446091) black band-diseased coral.
Sequence identity Isolate name (accession number) : Hyphomicrobium sulfonivorans (AY305006); Microbulbifer sp. MY05 (AY862188); Planctomycete strain 292 (AJ231182); Pseudomonas sp. GR2A19 (EF554921); Thiococcus sp. AT2204 (AJ401211); 2 unidentified Alphaproteobacteria isolates.