BIODEEP (EVK3-2000-00042) Second year Scientific Report

(April 1st 2002 – March 31st 2003)

Annex 1 to WP 4

Table 1 Media used on cruise in June 2002, for inoculation (UESSEX)

Anaerobic SRB 1 (modified after Brandt and Ingvorsen, Syst Appl Mic 20:366-373, 1997)

g/l
NaCl / 100
MgSO4* 7 H2O / 10
KCl / 5
CaCl2* 2 H2O / 0.2
NH4Cl / 2
KH2PO4 / 0.5
FeSO4* 7 H2O / 0.056
MnCl2* 2 H2O / 0.04
Citric acid (Na3Citrate) / 0.5 (0.76)
SL10 / 2 ml
Selenate + tungstate solution / 1 ml
Resazurin (0.5mg/ml) / 0.5 ml

add after autoclaving: 2 ml/l vitamin solution; 4 g/l NaHCO3 (autoclaved separately); 3mM Na2S; adjust pH to 7.0; use large inoculum (3-5% of sediment)

Table 2 Media used on cruise in June 2002, for inoculation (UESSEX) Anaerobic BfS

with sulfate
mM / g/l
NaCl / 3100 / 181
MgCl2*6H2O / 300 / 61
KCl / 100 / 7.5
CaCl2*2H2O / 15 / 2.2
Na2SO4 / 100 / 14.2
MgBr2*6H2O / 4 / 1.2
H3BO3 / 5 / 0.3
NaHCO3 / 30 / 2.5
ml/l
SL10 / 2
selenate + tungstate sol. / 1
resazurin (0.5mg/ml) / 0.5
SrCl2*6H2O (266.52, 10mM) / 0.05 / 5
LiCl (42.39, 100mM) / 0.075 / 0.75
NaF (70mM) / 0.07 / 1
NH4Cl (0.5M) / 0.5 / 1
KH2PO4 (0.1M) / 0.1 / 1
vitamin solution / 2
metasilicate (0.15M) / 0.15 / 1

final pH: 6.9 to 7.0 (adjust "brine" to pH 6.5 with NaOH before autoclaving)

NaHCO3 is saturated with CO2 and autoclaved separately in a tightly closed screw cap bottle

vitamins and metasilicate are added after autoclaving

reducing agents for media with sulfate: Na2S (100mM/2.5%, autoclaved, 0.1ml/10ml, final concentration 1mM) and/or dithionite (1%, sterile filtered, 0.1 ml/10ml)

reducing agents for media without sulfate: Na2S (as above) and cysteine (0.5g/l, sterile filtered) (will result nonetheless in small amounts of sulfate)

four different substrates for each of SRB-1 and Bfs

a) chitin, cellulose (100mg each per 100ml serum bottle)

b) volatile fatty acids (1mM acetate, 0.3mM propionate, 0.1mM butyrate, 0.1mM valerate, 0.05mM hexanoate, 0.05mM octanoate, 0.01mM nonate)

c) aromatic compounds (1mM benzoate, 1mM salicylate)

d) alcohols (1mM each ethanol, propanol, butanol, 2-propanol)

Table 3 Media used on cruise in June 2002, for inoculation (UESSEX)

Aerobic – Medium 1, modified Halobacteria medium (Halobacteriaceae Medium 3, p.415 Handbook of Microbiological Media, by Ronald M. Atlas; ed L. C. Parks, Boca Raton: CRC Press, c1993)

g/l
NaCl / 240
KCl / 7.5
MgCl2* 6 H2O / 5
MgSO4 (* 7 H2O) / 5 (10)
NH4Cl / 1
CaCl2* 2 H2O / 0.1
FeCl2* 6 H2O / 50 mg
(PIPES) / (3.4)
SL10 / 1 ml
KH2PO4, 100 mM / 1 ml

dissolve in 900ml water, adjust pH to 6.8, autoclave

Substrates: Glutamic acid, 20 ml of a 1M solution, pH 7.0 (filter sterilized); 5g yeast extract, 5g casaminoacids, dissolved in 80ml water, (filter sterilized)

Add substrates and 1 ml of vitamin solution to salt solution after autoclaving

Modifications: a) with PIPES b) without PIPES

Table 4 Media used on cruise in June 2002, for inoculation (UESSEX)

Aerobic – Medium 2, minimal medium for halophiles(Terry’s)

g/l
NaCl / 187
MgCl2* 6 H2O / 15.6
MgSO4* 7 H2O / 24
CaCl2* 2 H2O / 0.4
KCl / 4.8
NaBr / 0.6
NH4Cl / 1
H3BO3 / 0.3
FeSO4* 7 H2O / 50 mg
MnCl2* 4 H2O / 0.36 mg
KH2PO4 / 0.1
HEPES / 2.4
SL10 / 1 ml
SrCl2, 50 mM / 1 ml
LiCl, 100 mM / 1 ml
NaF, 70 mM / 1 ml
KH2PO4, 100 mM

dissolve in 970 ml water, adjust pH to 7.5, autoclave

autoclave separately: 0.25 g NaHCO3 in 20ml water (+ 0.5g/l soluble starch, 1ml/l glycerol for CPS), mix with salt solution after autoclaving

add 1ml/l vitamin solution after autoclaving

substrates: a) substrate mix, 10ml/l

b) CPS (0.5g/l bactopeptone, filter sterilized, 0.5g/l casaminoacids,

filter sterilized, 0.5g/l soluble starch, autoclaved, 1ml/l glycerol,

autoclaved)

iron precipitations

Table 5 Media used on cruise in June 2002, for inoculation (UESSEX)

Aerobic – Medium 3, modified Halococcus medium (DSM 146)

Solution B / g/l
NaCl / 200
MgSO4* 7 H2O / 10
NaNO3 / 1.7
KCl / 1.2
CaCl2* 2 H2O / 0.1
KH2PO4,100 mM / 1 ml
SL10 / 2 ml
(TRIS) / (1.2g)

dissolve in 850 ml water, adjust pH to 8.4, autoclave

solution A: 20g dried skimmed milk (TESCO) in 100ml water, autoclave at 112ºC for 20min

solution C: dissolve 2g bactopeptone, 2g glycerol in 50 ml water, filter sterilize

combine solutions after sterilization, add 1ml/l vitamin solution

modifications: a) with TRIS b) without TRIS

Table 6 Media used on cruise in June 2002, for inoculation (UESSEX)

Aerobic – Medium 4, modified Classical Halophile Medium (DSM 372)

g/l
NaCl / 200
MgSO4* 7 H2O / 20
CaCl2* 2 H2O / 0.2
Na3Citrate / 3
KCl / 2
FeSO4* 7 H2O / 50 mg
MnCl2* 4 H2O / 0.36 mg
SL10 / 1 ml
SrCl2, 50 mM / 1 ml
LiCl, 100 mM / 1 ml
NaF, 70 mM / 1 ml

dissolve in 840 ml water adjust pH to a) 7.6 b) 7.0, autoclave

add after autoclaving: 1ml/l vitamin solution, 10ml/l 1M glutamic acid, pH7.0, 5g yeast extract and 3.5g casamino acids dissolved in 150ml water and filter sterilized

Table7 Medium ONR7a (CNR)

solution 1 (700 ml)
NaCl / 22,79 g
Na2SO4 / 3,98 g
Taps / 1,3 g
KCl / 0,72 g
NH4Cl / 0,27 g
Na2HPO4 2H2O / 0,047 g
NaBr 1000 X (sol. Stock 3,1 g in 100 ml) / 1 ml
NaF 10000 X (sol. Stock 2,3 g in 100 ml) / 100 l
NaHCO3 1000 X (sol. Stock 3,1 g in 100 ml) / 1 ml
H3BO3 1000 X (sol. Stock 2,7 g in 100 ml) / 1 ml
Adjust pH to7,6
solution 2 (300 ml)
MgCl2 6H2O / 11,18 g
CaCl2 2H2O / 1,46 g
SrCl2 6H2O 1000 X (sol. Stock 2,4 g in 100 ml) / 1 ml

Prepare solution 1 and 2 and sterilize by autoclaving separately

After the solutions reach room temperature, combineand add 1ml of FeSO4 1000 X.

Table8DSMZ 193 derived medium (CNR DR 12)

Na2SO4 3 g/l, KH2PO4 0.2 g/l, NH4Cl 0.3 g/l, MgCl2.6H2O 1.3 g/l, KCl 0.5 g/l, CaCl2.2H2O 0.15 g/l, yeast extract 5 g/l, tryptone 5g/l, NaCl 50 g/l)

Table 9 Agar media used for isolation after enrichment cultures (CNR DR 12)

medium / Postgate / ZoBell / Halobacteria / Bannock
reference / DSMZ193 / DSMZ514 / DSMZ372 / Andrea Sass
Na / 2.6 M / 2.6 M / 2.6 M / 3.1 M
Cl / 2.6 M / 2.6 M / 2.6 M / 3.4 M
K / 6.7 mM / 8 mM / 27 mM / 100 mM
Mg / 6.4 mM / 62 mM / 81 mM / 300 mM
SO4 / 21 mM / 23 mM / 81 mM / 100 mM
Ca / 1 mM / 16 mM / 0 / 15 mM

All media contained (per liter) 15 g agar, 150 g NaCl (except Bannock : 181 g), 1 g chitin, cellulose, starch or casein or 10 ml olive oil, 0.2 g yeast extract ; pH = 7.

Samples collected during the cruise were stored at 4°C and inoculated at the laboratory in 100 ml bottles containing a substrate : 100 mg of chitin, cellulose, starch or casein, or 1 ml olive oil. Enrichment cultures were incubated at room temperature for 40 days; 10 ml of the cultures were re-inoculated after 10 days and 30 days in the same conditions. Enrichment cultures were plated on agar media (above) containing the substrate as carbon source and maintained at 25°C in aerobiosis or in a gaspak-generated anaerobic atmosphere, until colonies formation (1 month maximum). All isolates were obtained in aerobic conditions.

Table 10 Media used on cruise in June 2002, for inoculation (RUG)

Colourless sulfur bacteria medium (CSBM) (500 ml bottle = 600 ml)

3.7 mM NH4Cl

0.15 mM KH2PO4

1.53 mM CaCl2 · 2 H2O

2.68 mM KCl

1.0 mM MgCl2 · 6 H2O

Trace-elements (SL-12-B)

Na2-EDTA 3 mg · l-1

FeSO4 · 7 H2O1.1 mg · l-1

H3BO30.3 mg · l-1

CoCl2 · 4 H2O0.2 mg · l-1

MnCl2 · 4 H2O50 µg · l-1

ZnCl242 µg · l-1

NiCl2 · 6 H2O24 µg · l-1

Na2MoO4 · 2 H2O 18 µg · l-1

CuCl2 · 2 H2O2 µg · l-1

100 µM FeSO4

20 µg l-1 Cobalamine (vit. B12)

9.8 mM Na2CO3

4.0 mM Thiosulfate

pH adjusted to 7.0 with 1 M HCl

NaCl 3% or 10%

Table 11 Media used on cruise in June 2002, for inoculation (CoNISMA-Mi) – T1 Dethiosulfovibrio medium (Surkov et al, International Journal of systematic and evolutionary Microbiology (2001) 51,327-337)

(All the media have been prepared anoxically in an anaerobic glove box)

NH4Cl0,3 g/l

CaCl2.2H2O0,3 g/l

NaCl100 g/l

MgCl2-6H2O 3 g/l

KH2PO4 1 g/l

Sodium acetate 1 g/l

Yeast extract 1 g/l

Ferrous ammonium citrate 1 g/l

Elemental sulfur30 g/l

Resazurin (0,1%) 1 ml

Distilled water1000 ml

Adjust pH to 7 – 7,4

Table 12 Media used on cruise in June 2002, for inoculation (CoNISMA-Mi) – T2 Sulfate-reducing bacteria (modified from DSMZ media 193, 386, 503)

NaCl 100 g

MgCl2 x 6H2O 3 g

CaCl2 x 2 H2O0,15 g

Na2SO4 4 g

NH4Cl0,25 g

KH2PO40,2 g

KCl0,5 g

Resazurin (0,1%) 1 ml

Distilled Water 1000 ml

Trace element sol. 1 ml

Selenite tungstate sol. 1 ml

NaHCO3 sol. 30 ml

Vitamin mix 1 mlSee medium 4

Thiamin sol. 1 ml

Na2S sol 4 ml

Carbon sourcessee from 2.1 to 2.6

Adjust pH to 7 – 7,4

Carbon sources

T 2.12.1 Acetate and formiate 5 ml stock solution each.

T 2.22.2 Propionate and piruvate 5 ml stock solution each + palmitate 8 ml stock solution

T 2.32.3 Succinate, fumarate, malate pyruvate, 5ml stock solution each.

T 2.42.4 Ethanol 5 ml stock solution

T 2.52.5 Lactate 4 ml stock solution

T 2.62.6 Phenol + Catechol 2 ml stock solution

Sodium FormateStock solution 4 M

Sodium Acetate triidrato Stock solution 2 M

Sodium PropionateStock solution 0,6 M

Sodium Piruvate Stock solution 2 M

Sodium Palmitate Stock solution 0,1 M

Disodium Succinate hexahydrate Stock solution 0,5 M

Disodium Fumarate Stock solution 1 M

Disodium MalateStock solution 1 M

Ethanol11.6 ml of ethanol + 91.4 ml distilled water

Lactatelactate solution 50%

Phenol + Catechol2.4 mg phenol, 2.75 g catechol , 100 ml distilled water

Selenite tungstate solution

NaOH 0,4 g

Na2SeO3*5 H2O 6 mg

Na2WO4*2 H2O 8 mg

Distilled Water1000 ml

Trace Element solution (modified from Pfennig and Trüper, 1981)

Na-EDTA5,2g

FeSO4*7 H2O 12100 mg

H3BO330 mg

MnCl2*4H2O100 mg

CoCl2*6 H2O190 mg

NiCl2*6 H2O24 mg

CuCl2*2 H2O2 mg

ZnSO4*7 H2O144 mg

Na2MoO4*2 H2O36 mg

Distilled Water1000 ml

NaHCO3 solution (1.0 M)

84 g NaHCO3 are dissolved in distilled water to a final volume of 1 liter. The solution is dispensed in the required portions (30 ml for 1 liter of medium) into bottles, leaving approx. 1/3 as head space. Gassing of the head space with CO2 and saturation of the solution by repeated flushing and shaking is recommended. Bottles are tightly closed with rubber stoppers and autoclaved. Stoppers must be fixed before autoclaving with aluminum crimps or screw caps. Alternatively, bottles may be mounted between two parallel metal sheets that are held together by screws.

Thiamin solution

Thiamine chloride dihydrochloride10 mg

Sodium phosphate buffer 25 mM100 ml

pH3.4

Sulfide solution

Na2S*9 H2O 12 g/l

Distilled Water 250 ml

Table 13 Media used on cruise in June 2002, for inoculation (CoNISMA-Mi) – T3 Sulfur-reducing bacteria (modified from DSMZ media 193, 386, 503)

NaCl 100 g

MgCl2 x 6H2O3 g

CaCl2 x 2 H2O0,15 g

S elementar10 g

NH4Cl0,25 g

KH2PO40,2 g

KCl0,5 g

Resazurin (0,1%)1 ml

Distilled Water1000 ml

Trace element sol. 1 ml

Selenite tungstate sol. 1 ml

NaHCO3 sol.30 ml

Vitamin mix 1 mlSee medium 4

Thiamin sol. 1 ml

Vitamin B12 sol. 1 ml

Na2S sol 4 ml

Carbon source see fron T 3.1 to T 3.6. Adjust pH to 7 – 7,4

Carbon sources see medium T2

T3.13.1 Acetate and formiate 5 ml stock solution each.

T3.23.2 Propionate and piruvate 5 ml stock solution each + palmitate 8 ml stock solution

T3.33.3 Succinate, fumarate, malate piruvate, 5ml stock solution each.

T3.43.4 Ethanol 5 ml stock solution nolo

T3.53.5 Lactate 4 ml stock solution

T3.63.6 Phenol + Catechol 2 ml stock solution

Table 14Media used on cruise in June 2002, for inoculation (CoNISMA-Mi) – T4 Methanogenic Bacteria medium (Balch modified)

Mineral solution 150,00 ml

Mineral solution 250,00 ml

Basic mineral solution10,00 ml

Vitamin solution 5,00 ml

Sodium acetate2,50 g

Sodium formiate2,50 g

L-cys-HCl * H2O0,50 g

NaHCO32,50 g

Yeast extract1,00 g

Triptone1,00 g

NH4Cl1,00 g

FeSO4 * 7H2O (0,1%)1,00 ml

Resazurin (0,1%)1,00 ml

Na2S * 9H2O0,25 g

Distilled water to a final volume of 1 liter

Adjust pH to 7 – 7,4

Mineral solution 1

K2HPO4 6,0 g

Distilled water1 liter

Mineral solution 2

KH2PO4 6,00 g

(NH4)2SO4 6,00 g

NaCl 12,00 g

MgSO4* 7H2O 2,60 g

CaCl2 * 2H2O 0,16 g

Distilled water 1 liter

Basic mineral solution

Ac. nitrilotriacetico1,50 g

MgSO4* 7H2O3,00 g

MnSO4 * 2H2O0,50 g

NaCl 1,00 g

FeSO4 * 7H2O0,10 g

CoCl2 0,10 g

CaCl2 * 2H2O0,10 g

Na2WO4 * 2H2O0,10 g

Zn SO4 0,10 g

NiCl2 * 6H2O0,025 g

CuSO4 * 5H2O0,01 g

AlK(SO4)20,01 g

H3BO3 0,01 g

Na2MoO4 * 2H2O0,04 g

Na2SeO3 0,02 g

Distilled water1000 ml

pH 6,5-7

  • Vitamin solution

Biotin2,0mg

Folic acid2,0mg

Pyridoxine dihydrochloride 10,0mg

Thiamine hydrochloride5,0mg

Riboflavin5,0mg

Nicotinic acid5,0mg

Calcium D-L-pantothenate5,0mg

Vitamin B120,1mg

4-Aminobenzoic acid5,0mg

Lipoic acid5,0mg

Sodium ascorbate5,0mg

Coline chloride salt5,0mg

myo-Inositol5,0mg

Disdtilled water 500 ml

Table 15Media used on cruise in June 2002, for inoculation (CoNISMA-Mi) – T5 Anaerobe methanotrophic bacteria medium

Peptone 5 g

Meat extract 3 g

Na2SO4 4 g

NaCl 100 g

Distilled water 1000 ml

Methaneto replace head space

Adjust pH to 7.0.

Table 16Media used on cruise in June 2002, for inoculation (CoNISMA-Mi) – T6 Halanaerobiaceae medium (based on utilisation of a brine mix)

6.1 Yeast + Trypticase

Yeast2 g

Trypticase soy broth 2 g

Distilled water 666 ml

Brine mix 333 ml

6.2 Lactate

Lactate solution 2 ml (see medium 2)

Distilled water 666 ml

Brine mix333 ml

6.3 Propionate + Piruvate + Palmitate

Propionato2,5 ml Stock solution (see medium 2)

Piruvato 2,5 ml Stock solution (see medium 2)

Palmitato 4 ml Stock solution (see medium 2)

Distilled water 666 ml

Brine mix333 ml

Table 17Media used on cruise in June 2002, for inoculation (CoNISMA-Mi) – T7 Haloanaerobiuaceae medium (DSMZ 210 modified)

Glucose 2.5 g

NaCl130.0 g

MgSO4 x 7 H2O 8.8 g

KCl 1.0 g

Yeast extract 10.0 g

Trypticase (BBL) 10.0 g

Vitamin solution 5.0 ml (see medium 2)

Trace element (medium 144 DSMZ)10.0 ml

Distilled water 1000.0 ml

After autoclaving add from sterile solutions:

Thioglycolate-ascorbate 25.0 ml

Adjust pH to 7

Thioglycolate-ascorbate solution (reducing agent). Filter sterilize

Na-thioglycolate 0.3 g

Na-ascorbate 0.3 g

Distilled water 25.0 ml.

Trace element (medium 144 DSMZ)

Nitrilotriacetic acid 12.8 g

FeCl2 x 4 H2O 0.2 g

MnCl2 x 4 H2O0.1 g

CoCl2 x 6 H2O0.17 g

CaCl2 x 2 H2O0.1 g

ZnCl20.1 g

CuCl20.02 g

H3BO30.01 g

Na2MoO4 x 2 H2O0.01 g

NiCl2 x 6 H2O0.026 g

NaCl1 g

Na2SeO3 x 5 H2O0.02 g

Distilled water1000 ml

First, dissolve nitrilotriacetic acid and adjust to pH 6.5 with KOH.

Table 18 Media used on cruise in June 2002, for inoculation (CoNISMA-Mi) – T8 Geobacter medium (DSMZ 838)

KH2PO4 0.60 g

NH4Cl 0.30 g

MgSO4 x 7 H2O 0.50 g

CaCl2 x 2 H2O 0.10 g

Fe(III)-citrate (19% Fe) 10 g

NaHCO3, 10% w/v 1 ml

Trace element solution (See SL10) 0.01 ml

Selenite-tungstate solution (medium2) 0.01 ml

Na-acetate, 8% w/v 0.10 ml

Na-ascorbate, 8% w/v 0.10 ml

Distilled water 1000.00 ml

Dissolve ferric citrate by heating the water, cool to room temperature adjust the pH to 6.0 and add and dissolve the other ingredients. Adjust the medium pH to 6.8

Trace element solution SL-10:

Na2-EDTA 0.5 g/l

HCl (25%; 7.7 M) 10 ml

FeCl2 x 4 H2O 1.5 g

ZnCl2 70 mg

MnCl2 x 4 H2O 100 mg

H3BO3 6 mg

CoCl2 x 6 H2O 190 mg

CuCl2 x 2 H2O 2 mg

NiCl2 x 6 H2O 24 mg

Na2MoO4 x 2 H2O 36 mg

Distilled water 1000 ml

First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000 ml.

Table 19 Media used on cruise in June 2002, for inoculation (CoNISMA-Mi) – T9 Maintenance medium

Glucose 2.5 g

NaCl130.0 g

MgSO4 x 7 H2O 8,8 g

KCl 1.0 g

Yeast extract 5.0 g

Trypticase (BBL) 5.0 g

Vitamin solution5.0 ml (medium T4)

Basic mineral solution10.0 ml (medium T4)

Adjust pH to 7

Table 20 Number of isolates obtained from samples from 2002 cruise (CNR DR 12)

Sample* / substrate / total
chitin / cellulose / starch / casein / olive oil

L’Atalante

/ brine / 0 / 0 / 0 / 0 / 0 / 0
Bannock / interface SW/B / 0 / 2 / 2 / 0 / 0 / 4
p : 1
h : 1 / z : 2
brine / 0 / 0 / 0 / 0 / 0 / 0
Discovery / interface SW/B / 5 / 4 / 8 / 7 / 1 / 25
p : 1
z : 2
h : 2 / p : 2
z : 1
h : 1 / p : 2
z : 4
h : 2 / p : 3
z : 2
h : 2 / z : 1
brine / 0 / 0 / 0 / 0 / 0 / 0
Urania / brine / 0 / 0 / 0 / 0 / 0 / 0
interface B/HB / 0 / 1 / 0 / 3 / 0 / 4
z : 1 / p : 1
z : 1
h : 1
heavy brine / 0 / 0 / 0 / 0 / 0 / 0
mud pit / 0 / 0 / 0 / 0 / 0 / 0
total / 5 / 7 / 10 / 10 / 1 / 33

* interface SW/B = sea water / brine ; B/HB = brine / heavy brine.

Enrichment : 100 ml samples were inoculated in bottles containing 100 mg or 1 ml of substrate ; concentrated samples of Bannock brine (x6000), Discovery brine (x1640) and interface (x370), and Urania interface (x800) were inoculated to a final 4x concentration ; 10 ml of each culture was re-inoculated in 100 ml of the corresponding brine (+ substrate) after 10 and 30 days incubation.

Isolation : after 40 days, enrichment cultures were spread on agar plates (table 1) containing the substrate, and incubated at 25°C in aerobic or gaspak-generated anaerobic conditions until colonies formation (1 month maximum) ; all isolates were obtained in aerobiosis ; p = Postgate, z = ZoBell, h = Halobacteria medium ; no isolate was obtained on Bannock medium.

Table 21 Groups of restriction patterns of a 600bp PCR fragments amplified in a variable region of 16S rDNA, and molecular affiliation (strains isolated from 2001 samples) (CNR DR 12)

Groups of
restriction patterns / Strain n° / origin / Molecular affiliation
homology / aligned bases / genebank / genus
Unique patterns / 18
24
28
37
40 / Discovery sediment
Bannock brine
Discovery brine
Bannock interface
Urania interface / 97 %
97 %
99 % / 556/568
544/556
179/180 / ay121438
ay121438
aj244728 /

Halobacillus

Halobacillus

Marinobacter
Group A / 29
34
36
39
46
47 / Urania interface
Bannock interface
Bannock interface
Bannock interface
Bannock interface
Bannock interface / 98 %
100 % / 274/278
566/566 / y16735
y16735 /

Marinobacter

Marinobacter
Group B / 30
31
32
38 / Urania interface
Discovery interface
Discovery interface
Bannock interface / 100 %
98 % / 539/540
393/399 / ab042501
af212217 / Halomonas
Halomonas
Group C / 63
64 / Discovery sediment
Discovery sediment / 97 %
97 % / 323/334
289/295 / aj291752
aj291752 /

Halobacillus

Halobacillus
Group D / 1
33
35 / Urania interface
Bannock interface
Bannock interface / 98 %
97 % / 345/349
264/272 / af529060
af297958 /

Alteromonas

Pseudoalteromonas

PCR amplification was performed using primers GM5F and 907R (Teske et al., 1998 - Appl. Env.Microbiol. 64: 2943-2951). Sequencing was performed by Dr Sara Borin with primer 926f.

Table 22 Phylogenetic affiliation of strains (CNR)

# / Complete
Sequence / Partial
Sequence / Affiliation
320 / X / Idiomarina l
323 / X / Marinobacter aqueoli
326 / X / Marinobacter hydrocarbonoclasticus
327 / X / Marinobacter hydrocarbonoclasticus
328 / X / Pseudomonas pseudoalcaligenes
330 / X / Marinobacter aqueoli
333 / X / Pseudomonas pseudoalcaligenes
339 / X / Halomonas variabilis
340 / X / Alteromonas sp.
345 / X / Marinobacter hydrocarbonoclasticus
350 / X / Marinobacter hydrocarbonoclasticus
353 / X / Alteromonas sp.
354 / X / Marinobacter aqueoli
356 / X / Idiomarina l
357 / X / Alcanivorax Sp
358 / X / Alcanivorax venusti
359 / X / Alcanivorax venusti
363 / X / Marinobacter aqueoli
364 / X / Halomonas variabilis
365 / X / Halomonas variabilis
367 / X / Halomonas variabilis
368 / X / Alcanivorax sp.
369 / X / Halomonas variabilis
370 / X / Marinobacter aqueoli
373 / X / Pseudoalteromonas tarctica
374l / X / Marinobacter hydrocarbonoclasticus
374b / X / Marinobacter hydrocarbonoclasticus
348b / X / Idiomarina l
348g / X / Marinobacter aqueoli
351b / X / Marinobacter aqueoli
351g / X / Marinobacter aqueoli

Figure 1 REP fingerprinting analysis; dendrograms of electrophoresis and 16SrDNA-based affiliation (CNR)

Figure2 Dendrogram from Repetitive Extragenic Palyndromic - PCR (rep-PCR) fingerprint of isolated strains. Strains reported with the same colour belong to the most abundant ITS-groups.rep-PCR was performed using the primer BOX A1R, which anneals to repetitive regions scattered throughout the genome exhibiting homologous sequences.This dendrogram illustrates the similarity of strains based on cluster analysis of a matrix of data reporting the presence/absence of every band resulting from rep-PCR (CoNISMA-Mi)

Figure 3 DGGE profile from Discovery seawater-brine interface (lane 1) and cultivated strains from the same sample (CoNISMA-Mi)


Table 23 Eubacteria and Archaea cell quantification by real-time PCR (CoNISMA-Mi)

Urania / Bannock
W/B interface / Brine / W/B interface / Brine
Archaea (cells/ml) / 9 104 / 3 103 / 2 105 / n.d
Eubacteria (cells/ml) / 3 106 / 2 105 / 1 106 / n.d

n.d.: not determined

Table 24 Enrichments from the 2002 Cruise (CoNISMA-Mi)

mixed cultures / isolated strains / Identified strains / basin / sample / medium / C sorce / e- donors / e- acceptors / other / Medium for culrivation of
1 / B / BS / 1 / Sodium acetate; Yeast extract; Ferrous ammonium citrate / Sodium acetate; Ferrous ammonium citrate / Elementar sulfur / Sulfur-reducing bacteria
1 / 1UB02 / U / B
2UB02 / Haloanaerobium prevalens / U / B
3UB02 / Haloanaerobium prevalens / U / B
1 / B / BS / 2.02 / Propionate; piruvate; palmitate / Propionate; piruvate; palmitate / Na2SO4 / Sulfate-reducing bacteria
1 / A / BS / 2.03 / Succinate, fumarate, malate piruvate / Succinate, fumarate, malate piruvate / Na2SO4 / Sulfate-reducing bacteria
1 / B / BS / 2.04 / Ethanol / Ethanol / Na2SO4 / Sulfate-reducing bacteria
1 / A / BS
1 / B / BS / 2.05 / Lactate / Lactate / Na2SO4 / Sulfate-reducing bacteria
1 / A / BS / 3.01 / Acetate, formiate / Acetate, formiate / Elemental sulfur
Elemental sulfur / Sulfur-reducing bacteria
1 / A / BS / 3.02 / Propionate; piruvate; palmitate / Propionate; piruvate; palmitate / Elemental sulfur / Sulfur-reducing bacteria
1 / B / BS / 3.03 / Succinate, fumarate, malate piruvate / Succinate, fumarate, malate piruvate / Elemental sulfur / Sulfur-reducing bacteria
1 / A / BS
1 / B / BS / 3.04 / Ethanol / Ethanol / Elemental sulfur / Sulfur-reducing bacteria
1 / A / BS
1 / B / BS / 3.05 / Lactate / Lactate / Elemental sulfur / Sulfur-reducing bacteria
1 / B / BS / 3.06 / Phenol, Catechol / Phenol, Catechol / Elemental sulfur / Sulfur-reducing bacteria
1 / U / B
1 / 1BBS02 / B / BS / 4 / CO2, Sodium acetate, Sodium formiate / Sodium acetate, Sodium formiate / CO2 / Yeast extract, Triptone / Methane bacteria
6BBS02 / Uncultured bacterium / B / BS
7BBS02 / Thermobacillus barrensis / B / BS
1 / 7UB02 / U / B
8UB02 / U / B
6UB02 / Haloanaerobium prevalens / U / B
1 / 1ABS02 / Bacillus pantotenticus / A / BS
1 / 4DBS02 / Bacillus licheniformis / D / BS
1 / B / BS / 5 / CH4 / CH4 / Na2SO4 / Peptone, Meat extract / Anaerobic methano-trophic bacteria
1 / A / B
1 / U / BS
1 / 2ABS02 / A / BS
1 / 9DBS02 / D / BS
5DBS02 / Bacillus licheniformis / D / BS
6DBS02 / Bacillus licheniformis / D / BS
1 / 1DB02 / Haloanaerobium prevalens / D / B
1 / 10UB02 / U / B
1 / B / BS / 6.01 / Yeast extract, Trypticase soy broth / Brine / Haloanaerobiaceae
1 / A / B
1 / A / BS
1 / D / BS

Table 24 continued

mixed cultures / isolated strains / Identified strains / basin / sample / medium / C sorce / e- donors / e- acceptors / other / Medium for culrivation of
1 / B / BS / 6.02 / Lactate / Brine / Haloanaerobiaceae
1 / A / B
1 / A / BS
1 / B / BS / 6.03 / Propionate, Piruvate, Palmitate / Brine / Haloanaerobiaceae
1 / A / BS
1 / D / BS
1 / A / BS / 7 / Glucose, Yeast extract, Trypticase / Haloanaerobiaceae
1 / D / B
1 / 9UB02 / U / B
1 / 10BBS02 / Bacillus pantotenticus / B / BS
11BBS02 / Bacillus pantotenticus / B / BS
12BBS02 / Bacillus pantotenticus / B / BS
2BBS02 / Bacillus pantotenticus / B / BS
1 / A / BS / 8 / Citrate / Citrate / Fe3+ / Geobacter
1 / D / BS
1 / U / BS
1 / 4BBS02 / B / BS
1 / B / D / 9 / carbon dioxide / Sodium thiosulfate / oxygen / Sulfur oxidant
1 / U / BS / 10 / Methane / Methane / Sodium sulfate / Methane oxidant


Figure 4 Survival of cells inoculated in the brines (A, L’Atalante; B, Bannock) (CoNISMA-Mi)

Figure 5 Survival of cells inoculated in the brines (D, Discovery; U, Urania) (CoNISMA-Mi)


BIODEEP (EVK3-2000-00042) Second year Scientific Report

(April 1st 2002 – March 31st 2003)

Table 25 Brine Samples Inoculated June/July 2002 (UESSEX)

Samples

/ Date / Time / Reference / Niskin / Conc fact /

Date inoc An

/

SRB-cell

/ BFS-cell / SRB-fa / BFS-fa / SRB-aro / BFS-aro / SRB-alc / BFS-alc / Date inoc Aer / 1a / 1b / 2a / 2b / 3a / 3b / 4a / 4b
UW- B1 / 6/6 14:10 / BD 05 CT
#20 / 7/6 / - / - / + / +* / + / + / +* / +
UW- B1 conc / 6/6 / BD 04/05 CT / 2200
A-B / 7/6 23:00 / BD 08 CT #2 / 11/7 / - / - / - / - / - / 8/6 / - / - / +* / - / - / +R / - / -
A-B conc / 6/6 / BD 08/09 CT / 8400
D-B / 9/6
04:00 / BD 13 CT #21 / 9/6 / - / - / - / - / - / - / - / -
D-B conc / 9/6 / BD 12/13 CT / 2700
UE-interfaces conc / 9/6 / BD 16 CT / 800 / 11/7 / - / - / + / +* / + / + / + / +*
D-interface conc / 10/6 / BD CT / ?
B-B / 14/6 / BD 29 CT#6 / 11/7 / - / - / - / - / - / 11/7 / - / - / - / - / - / - / - / -
B-B conc / 14/6 / BD 29 CT / 6000

UW-B1 was flushed for 5 hours before inoculation (this may have served to remove toxic H2S and explain the good growth in aerobic samples

On 11/7 in aerobic enrichments (except UW-B1) the butyl stoppers were punctured with a large gauge needle in which cotton wool had been stuffed into the top.

+ = positive signs of growth (final check 20/5/03). It was sometimes difficult to tell for sediment samples, and so if in doubt they have been marked – (negative). * = sample was spread onto agar plate of equivalent composition. Blank = no inoculation.

Table 26 Sediment Samples Inoculated June/July 2002 (UESSEX)

Samples

/ Date/ Time / Reference / Region /

Date inoc An

/

SRB-cell

/ BFS-cell / SRB-fa / BFS-fa / SRB-aro / BFS-aro / SRB-alc / BFS-alc / Date inoc Aer / 1a / 1b / 2a / 2b / 3a / 3b / 4a / 4b
UW B1-S / 6/6 21:00 / BD ? BC / 0-4 cm / 11/7 / + / + / - / - / - / + / + / +
UE B1 basalt / 8/6 / BD 01 ST3
UE B1 rhyolite / 8/6 / BD 01 ST3
D-S / 10/6 23:00 / BD ? MC / 0-8 cm / 10/7 / - / - / - / - / - / - / - / - / 11/7 / - / + / - / + / + / + / + / +*
UE-S / 11/6
20:45 / BD 21 MC / 0-10 cm / 10/7 / - / - / - / - / - / - / - / - / 11/7 / - / - / - / +* / - / + / - / +
UE-S / 11/6
20:45 / BD 21 MC / 52-58 cm
A-S gypsum / 13/6 10:45 / BD 26 BC / 10/7 / -
A-S / 13/6 10:45 / BD 26 BC / 26-35 cm top / 10/7 / - / - / - / - / - / - / - / - / 11/7 / - / + / - / +* / + / + / + / +*
A-S / 13/6 10:45 / BD 26 BC / 50-55 cm bott
UW-MP / 16/6 14:00 / BD 36 CT / 10/7 / - / - / - / - / - / - / - / - / 11/7 / - / - / - / - / - / - / - / -
UW-MP gypsum / 16/6 14:00 / BD 36 CT
B-S gypsum / 18/6 / BD 40 CT
B-S / 18/6 / BD 41 CT / top / 11/7 / - / + / - / +* / + / + / + / +*
B-S / 18/6 / BD 41 CT / pellicle

+ = positive signs of growth (final check 20/5/03). It was sometimes difficult to tell for sediment samples, and so if in doubt they have been marked – (negative). * = sample was spread onto agar plate of equivalent composition. Blank = no inoculation.

Table 27 Summary of Phylotypes (All partners) See Annex 2 for details

Phylogenetic group / AI / AB / AS / BI / BB / BS / UI / UB / US / DI / DB / DS / SW / ? / Total
Archaea - Haloarchaea
/ 1 / 0 / 1 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 2
Archaea - Methanosarcinales / 0 / 0 / 4 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 4
Firmicutes - Bacillales / 2 / 0 / 42 / 1 / 0 / 42 / 0 / 2 / 29 / 7 / 1 / 31 / 1 / 0 / 158
Firmicutes - Clostridiales / 0 / 0 / 17 / 0 / 0 / 6 / 0 / 0 / 1 / 0 / 0 / 0 / 0 / 0 / 24
Firmicutes - Lactobacillales / 0 / 0 / 0 / 0 / 0 / 1 / 1 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 2
Alpha Proteobacteria / 1 / 0 / 1 / 2 / 0 / 1 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 5
Gamma Proteobacteria - unknown / 0 / 0 / 0 / 0 / 0 / 1 / 1 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 2
Gamma Proteobacteria - Alteromonadaceae / 9 / 0 / 0 / 21 / 0 / 0 / 3 / 0 / 5 / 4 / 1 / 1 / 0 / 1 / 45
Gamma Proteobacteria - Chromatiaceae / 0 / 0 / 0 / 3 / 0 / 0 / 14 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 17
Gamma Proteobacteria - Enterobacteriales / 0 / 0 / 0 / 2 / 0 / 0 / 1 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 3
Gamma Proteobacteria - Halomonadaceae / 2 / 0 / 2 / 4 / 0 / 7 / 1 / 4 / 3 / 7 / 0 / 1 / 0 / 0 / 31
Gamma Proteobacteria - Idiomarina / 1 / 0 / 0 / 3 / 0 / 0 / 4 / 0 / 0 / 0 / 0 / 0 / 0 / 1 / 9
Gamma Proteobacteria - Marinobacter-Alcanivorax / 4 / 1 / 3 / 40 / 1 / 1 / 2 / 1 / 1 / 0 / 0 / 0 / 4 / 5 / 63
Gamma Proteobacteria - Pseudomonadaceae / 1 / 0 / 0 / 1 / 0 / 6 / 1 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 9
Gamma Proteobacteria - Vibrionales / 3 / 0 / 0 / 2 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 5
Epsilon Proteobacteria / 0 / 0 / 0 / 1 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 1
Actinobacteria
/ 0 / 0 / 2 / 0 / 0 / 2 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 4
Bacteroidetes / 0 / 0 / 0 / 2 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 2
Halanaerobiales / 5 / 0 / 6 / 2 / 0 / 0 / 3 / 3 / 0 / 0 / 1 / 1 / 0 / 0 / 21
Total / 29 / 1 / 78 / 84 / 1 / 67 / 31 / 10 / 39 / 18 / 3 / 34 / 5 / 7 / 407

? = 7 strains from CNR awaiting provenance

bold font indicates obligate anaerobes

BIODEEP (EVK3-2000-00042) Second year Scientific Report

(April 1st 2002 – March 31st 2003)

Figure 6 Phylogeny of Bacillales isolates (UESSEX)


Figure 7 Phylogeny of isolates related to Bacteroidetes and Epsilon-Proteobacteria(UESSEX)


Figure 8 Phylogeny of Isolates in the Clostridiales(UESSEX)


Figure 9 Phylogeny of Isolates in the Halanaerobiales (UESSEX)