Effects of seawater ozonation on biofilm development in aquaculture tanks

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. To assess the effects of ozonation on biofilm development and composition in small-scale aquaculture tanks containing the brine shrimp Artemia (commonly used as live feed in larval rearing of various species). Ozonation of seawater alters water chemistry, changing selection pressures on bacterial populations, for bacteria surviving the treatment as well as bacteria introduced to aquaculture tanks from other sources such as live feed.Microbial biofilms in aquaculture tanks represent a reservoir for opportunistic bacterial pathogens, and procedures to control formation and bacterial composition of biofilms are important for the development of commercially viable aquaculture industries.

本研究采用显微镜观测与分子生物学技术，对接收臭氧消毒水与非臭氧消毒水的养殖槽内生物被膜（biofilm）的发育过程开展了为期44天的监测。本次检测到的生物被膜内细菌类群包括：γ-变形菌纲（Gammaproteobacteria）、α-变形菌纲（Alphaproteobacteria）、β-变形菌纲（Betaproteobacteria）、浮霉菌目（Planctomycetales），以及拟杆菌门（Bacteroidetes）下的噬纤维菌/黄杆菌类群（Cytophaga/Flavobacterium branch）。

变性梯度凝胶电泳（Denaturing Gradient Gel Electrophoresis, DGGE）测序与荧光原位杂交（Fluorescence In Situ Hybridization, FISH）技术用于细菌菌种鉴定。针对每种实验处理组，将八孔载玻片（用于荧光原位杂交实验）与普通载玻片（用于变性梯度凝胶电泳实验）置入小型水产养殖槽中；养殖槽供应经过过滤的臭氧消毒海水或非臭氧消毒海水，水流恒定为1.0~1.2 L·min⁻¹，并向槽内投放卤虫（Artemia）。此后，每日通过移除残留卤虫并添加新鲜采收的卤虫完成种群更换；同时通过虹吸法清除每个养殖槽底部的死亡卤虫与颗粒物。实验全程监测卤虫的死亡率。

在首次向养殖槽投放卤虫后的1小时（第0天）与24小时（第1天）分别采集八孔载玻片。后续采样每隔2天进行一次（第3、5、7、9、11、14、16、18、20天），之后改为每周采样一次（第28、36、44天）。

所用探针及其特异性如下：EUB338b（靶向绝大多数细菌）；EUB338-IIb（靶向额外细菌类群——浮霉菌目）；EUB338-IIIb IIb（靶向额外细菌类群——疣微菌目）；NON338（EUB338的反义探针）；ALF1B（靶向α-变形菌纲及部分其他细菌）；BET42ad（靶向β-变形菌纲）；GAM42ad（靶向γ-变形菌纲）；CF319a（靶向拟杆菌门下的噬纤维菌/黄杆菌类群）；GV（靶向弧菌属（Vibrio））；PLA886d、e与PLA46e（靶向浮霉菌目）；G123d（靶向丝硫菌属（Thiothrix））。

从生物被膜变性梯度凝胶电泳图谱切取的条带中获取的序列的系统发育归属如下：
序列名称（登录号） 来源环境：克隆b1uc29（EF206937） 白化珊瑚；克隆A714001（AY907796） 阿拉伯海超微型浮游生物；克隆JTB23（AB015248） 深海冷泉沉积物；嗜硫红微菌（Rhodovulum sulfidophilum）ATCC 35886（DQ342323） 潮间带泥滩；克隆b2ub58（EF206984） 白化珊瑚；玫瑰杆菌属（Roseobacter sp.）49Xb1（EU090129） 莹虾（Nyctiphanes simplex）胃内；克隆BBD（DQ446091） 黑带病珊瑚。
序列同源性 分离株名称（登录号）：噬硫生丝微菌（Hyphomicrobium sulfonivorans）（AY305006）；微泡菌属（Microbulbifer sp.）MY05（AY862188）；浮霉菌菌株292（AJ231182）；假单胞菌属（Pseudomonas sp.）GR2A19（EF554921）；硫球菌属（Thiococcus sp.）AT2204（AJ401211）；2株未鉴定的α-变形菌纲分离株。

本研究旨在评估臭氧消毒对含有卤虫（Artemia，常作为多种水产动物幼体培育的活饵料）的小型水产养殖槽内生物被膜发育与群落组成的影响。海水臭氧消毒会改变水体化学性质，进而改变细菌种群的选择压力——既作用于经消毒处理后存活的细菌，也作用于从活饵料等其他途径引入养殖槽的细菌。水产养殖槽内的微生物被膜是机会性致病细菌的储存库，因此调控生物被膜的形成与细菌群落组成，对于推动水产养殖业实现商业化可持续发展具有重要意义。