Effect of Mid-Ocean Exchange of Ballast Water on Bacterial Community in Ballast Tanks

Transcription

1 Effect of Mid-Ocean Exchange of Ballast Water on Bacterial Community in Ballast Tanks 58,098 GT Length(O.A.) m Length(PP) m Breadth 43.00m Depth 20.50m Akiko Tomaru 1, Yasuwo Fukuyo 1, Masanobu Kawachi 2 and Hiroshi Kawai 3 The University of Tokyo Asian Natural Environmental Science Center 1, National Institute for Environmental Studies 2, Kobe University 3

2 Ballast Water Problems Cargo landing port = Uptaking Ballast water Ballast water transfer alien species Natural ecosystem disturbance Economic Cargo loading port loss = Discharging ballast water Human illness etc. Ballast Water Stoways

3 Ballast Water Problems Ballast Water Stoways International maritime organization (IMO) adopted so-called Ballast water management convention ( Feb. 2004)

4 Purpose of this study To observe the change of bacteria community in ballast tanks before and after the mid-ocean exchange, and also during the voyage.

5 Voyages used for Observation Route: Japan (Uptaking ballast water) Observation: 58,098 GT Length(O.A.) m Length(PP) m Breadth 43.00m Depth 20.50m Working men Mid-ocean exchange Australia (Discharging ballast water) Winter; 2nd - 18th December, 2004 Summer; 20th, June - 2nd, July, 2005

6 Sampling from Ballast Tanks No.2 WBT P 2,809 m 3 No.1 WBT P No.2 WBT P No.3 WBT P No.4 WBT P No.5 WBT P No.6 TST P No.7 TST P WWT P No.7 TST P 760 m 3 FPT No.4 CH No.6 DBT No.1 WBT S No.2 WBT S No.3 WBT S No.4 WBT S No.5 WBT S No.6 TST S No.7 TST S No.7 DBT WWT S #2P #7P APT

7 Ballast Water Sampling Surface (Manhole) Bottom (Sounding pipe)

8 Quantitative Direct count (Total count) : No distinction between live and dead cells Qualitative Denaturing Gradient Gel Electrophoresis (DGGE) : All bacteria Plate Count (Live count) : Live cells

9 Direct Count (Total count) Preparation Fixation, Stain Count Epifluorescen ce Microscopy Filtration Keep on -20 C Bright blue-white cells DAPI x1000 On board Laboratory

10 Plate Count (Live count) Inoculation Count Culture: Room temperature, 2days (non-selective) Marine Agar (Difco) CFU = Colony forming unit

11 DGGE (Denaturing Gradient Gel Electrophoresis) PCR amplify Primer with GC-richi sequenc (F341GC,R907) Preparation (onboard) 5μm 0.2μm filtration Keep under -20 C DNA extraction Electrophoresis Denatured (urea and formamige) polyacrylamide (acrylamide/bisacrlamide) gel Visualization by Stain Analysis of DGGE patterns

12 Bacteria Number - Winter Before Direct Count (cells/ml) DC-PC Mid-ocean Re-ballast exchange After DC-SW DC-#7S DC-#7B DC-#2S DC-#2B PC-SW PC-#7S PC-#7B PC-#2S PC-#2B The numbers by the direct counts and the plate count (bottom) significantly decreased. DC, Plate Count 10 3 Surface; (CFU/ml) W = 0, p = 0.002: Bottom; W = 0, p = PC, Surface; W = 12, p = 0.142: Bottom; W = 4, p = (#7PBWT; Wilcoxson test) Day

13 Bacteria Number - Summer Before Direct Count (cells/ml) DC-PC Mid-ocean Re-ballast exchange After DC-SW DC-#7S DC-#7B PC-SW PC-#7S PC-#7B Only the numbers Plate by Count the direct (CFU/ml) count significantly decreased. DC, Surface; W = 24, p = 0.010: Bottom; W = 23, p = PC, Surface; 4 W = 12, p = 1.0 : Bottom; W = 13, p = (Wilcoxon test) Day

14 Part of regulation D-2 Regulation D-2 Ballast Water Performance Standard Indicator microbes, as a human health standard, shall include:.1 Toxicogenic Vibrio cholerae (O1 and O139) with less than 1 colony forming unit (cfu) per 100 millilitres or less than 1 cfu 1 gram (wet weight) zooplankton samples;.2 Escherichia coli less than 250 cfu per 100 millilitres;.3 Intestinal Enterococci less than 100 cfu per 100 millilitres. Balast water management convention International Maritime Organization (IMO); February, 2004

15 Culture Media Non-selective Media Selective Media Marine Agar (Difco) R2A (Difco) Heterotrophic bacteria X-Gal Agar (Nissui) TCBS Agar (Nissui) Marine bacteria Fresh water bacteria Coliforms Group of (Blue color colonies) Vibrio cholerae (Yellow color colonies)

16 (CFU/ml) Plate count (Live bacteria) - Summer Plate Count Mid-ocean Re-ballast exchange Coliforms Marine 1. The mid-ocean exchange has 10 4 no effect to decease live bacteria. 2. The live bacteria in coastal waters Fresh Water 10 were removed 2 by the mid-ocean exchange. Vibrio cholerae group Day SW-Marine #7S-Marine #7B-Marine SW-FW #7S-FW #7B-FW SW-Coliforms #7S-Coliforms #7B-Coliforms SW-V.cholerae #7S-V.cholerae #7B-V.cholerae

17 DGGE-Winter SW01 BW01S BW03S BW03B BW05S BW05B SW02 BW07S BW07B BW09S BW09B BW14S BW14B BW15S BW15B BW16S BW16B AU07 AU08 Marker ( 100bp) SW (Black): Uptaking sea water sample; #1, Japan port area; #2, Mid-ocean water. BW: Ballast water; S, Surface; B, bottom; #, days after charge; Red, Before the mid-ocean exchange (#7tank); Blue, After the mid-ocean exchange (#7tank); Green, After the mid-ocean exchange (#2tank) % 70V, 14hr Classification methods Cluster Analysis Ordimation methods Nonmetric Multidimentional Scaliing(MDS) BW(surface&bottom) : SW01, soma; SW02, ocean; S, surface; B, bottom; BW01-05, before exchanged ballast water; BW07-16, after exchanged ballast water; BW01-15, #7P BWT; BW16, #2P BWT; AU: Australia

18 Cluster Analysis - Winter Surface Bottom Bacterial floras were different before and after the mid-ocean exchange. BW10B BW9B BW13B BW11B BW12B BW8B BW7B SW2 AU7 BW16B BW11B BW13B BW4B BW3B SW1 BW5B BW6B BW13S BW12S BW14S BW15S BW11S BW8S BW10S BW7S BW16S BW11S AU7 SW2 BW2S BW1S SW1 BW4S BW3S BW6S BW5S Before After Before After

19 BW1S BW2S SW1 BW3S BW5S BW4S BW6S Before-#7 Surface Mid-ocean exchange MDS - Winter BW16S BW11S Bottom #2 #2 BW11B BW16B BW15B BW15S SW1 BW14S BW3B BW13S BW5B AU7 BW4B BW6B BW13B BW12S BW12B after the mid-ocean exchange. BW14B BW10B Before-#7 BW8B BW10S BW7B BW8S BW7S BW9S BW9B SW2 SW2 formed After-#7 in the tanks No.2 and After-#7 AU7 Mid-ocean exchange 1. Bacterial floras were different 2. Different bacteria floras were No.7.

20 Summary 1. After the mid-ocean exchange, total bacteria numbers significantly decreased. 2. Live bacteria number did not change significantly after the exchange, but live bacteria in the coastal waters were removed by the exchange. 3. Bacteria flora changed gradually in ballast tanks day by day, and became drastically different by the exchange.