Interactions of microalgae andInteractions of microalgae and microbial communities present in

microalgae cultures and larval rearing systemssystems

Pavlos Makridis Jose Pintado Tania Fereira FoteiniPavlos Makridis, Jose Pintado, Tania Fereira, Foteini Kokou, Kostas Tsigenopoulos, Pascal Divanach

Hellenic Center for Marine Research, Iraklio; GreeceInstituto de Investigacións Mariñas, CSIC, Vigo, Spain

Bacteria in microalgae cultures?

Greenwater = microalgae + bacteria

Bacteria in microalgae cultures may have a positiveBacteria in microalgae cultures may have a positiveinfluence for the larvae

Three series of experiments:

1. bacterial communities in cultures of Chlorella minutissima

B t i l iti i fl d bBacterial communities influenced by:

• algal species cultured• cultivation system• phase of the culture (lag, log, stationary, death)p ( g, g, y, )• state of culture• other factors (use of antibiotics contaminations• other factors (use of antibiotics, contaminations etc)

Small bottles (0.5-5 L) autoclaved waterSmall scale

Large bottles (5-20 L) chlorine

Sleeves (100-400 L) chlorine

PBR (>1 m3) chlorineLarge scale

200 L sleeves200 L sleeves

Natural light conditionsg

Sampling for microbiological analysis

2, 5, 9, 14 days after inoculation2, 5, 9, 14 days after inoculation

nitrofurantoin (10 mg L-1) control

3200 g 10 min M65 TCBS3200 g, 10 min M65, TCBS

SPN

WA

CFU - Sleeves X1 and X2 cultured in M653E+05

per

cel

l9E+06

1E+07

U, p

er m

l

2E+05

CFU

,

7E+06

8E+06CFU

2E+05

5E+06

6E+06

1E+053E+06

4E+06

5E 06

1E+06

2E+06

3E+06

0E+00 0E+00

1E+06

2_X1 5_X25_X12_X2 9_X29_X1 14_X214_X1

X1_WA_cell X1_SPN_cell X2_WA_cell X2_SPN_cellX1_WA_ml X1_SPN_ml X2_WA_ml X2_SPN_ml

CFU - Sleeves A1 and A2 cultured in M653E+05

per

cel

l9E+06

1E+07

per m

l

CFU

,

7E+06

8E+06CFU

,

2E+05

5E+06

6E+06

7E+06

1E+054E+06

5E+06

1E+05

2E+06

3E+06

0E+00 0E+00

1E+06

2 A1 5 A25 A12 A2 9 A29 A1 14 A1 14 A2A1_WA_cell A1_SPN_cell A2_WA_cell A2_SPN_cellA1_WA_ml A1_SPN_ml A2_WA_ml A2_SPN_ml

2 A1 5_A25_A12 A2 9_A29_A1 14_A1 14_A2

7080

506070

cter

ial

304050

er o

f ba

stra

ins

102030

Num

be

00

2WA 2S 5WA 5S 9WA 9S 14WA 14S+gram- gram +

Strain BLAST result Homology %2SA01 Pseudomonas sp. D6-9 99

2SA02 Stenotrophomonas maltophilia isolate SMJR2 100

2SA03 Vibrio sp. SMB7 99

2AX04 Gamma proteobacterium 12IX/A01/168 99

2AX05 Muricauda aquimarina strain SW-72 982AX05 Muricauda aquimarina strain SW 72 98

2SX06 Loktanella rosea 995AA07 Roseobacter sp. YS-57 995AX08 Halomonas sp. B12 995SX09 Pseudoalteromonas sp es32 99

5SX10 Maribacter goseongensis strain IS14 99

5SX11 Paracoccus marcusii 1009SX12 Arenibacter sp. CC10 99

14AA13 Erythrobacter sp. SMB19 99

14AA14 Maribacter goseongensis strain IS14 99

14AA15 Halomonas sp. B12 99

14AA16 Gamma proteobacterium 12IX/A01/168 99

14AA17 Marinobacter sp. NT N31 9914AA18 Loktanella rosea 99

14SA19 Flexibacter aggregans strain BSs20185 99

14AX20 Roseobacter sp. YS-57 99

counts in TCBS

Densities CFU per ml CFU per cell

Extract DAI 2 5 9 14 2 5 9 14

A1 10 250 190 100 0 2 1 0

WAA2 0 0 100 120 0 0 0 0

X1 0 0 30 0 0 0 0 0

X2 0 20 20 0 0 0 1 0X2 0 20 20 0 0 0 1 0

A1 300 100 270 630 10 1 1 1

A2 270 0 430 440 7 0 1 1SPN

A2 270 0 430 440 7 0 1 1

X1 180 40 50 250 7 1 0 1

X2 230 20 70 0 8 0 0 0X2 230 20 70 0 8 0 0 0

Summary of findings

1. Low numbers of presumptive Vibrio

2. Counts in M65 higher in whole algae samples

than in SPN sampesp

3. The numbers of presumptive Vibrios and opportunistists

in whole algae was lower than in SPNg

4. The numbers of presumptive Vibrio in cultures

added antibiotic was higher than in the controladded antibiotic was higher than in the control

cultures

M65 TCBS3200 g, 10 min

M65, TCBS

SPN

WA

Three series of experiments:Three series of experiments:

1 b t i l iti i lt f1. bacterial communities in cultures of Chlorella minutissima

2. Effect of five microalgae species on g pVibrio bacteria

• Incubation of each bacterial strain separately in axenicIncubation of each bacterial strain separately in axenic microalgae culture

• A control for each trial with bacteria inoculated in sterile medium

• Natural light conditionsNatural light conditions

• Initial bacterial concentration 104 cells per mLInitial bacterial concentration 10 cells per mL

Light Dark

Conclusions

Vibrio bacteria grew well in microalgae medium in the absence of microalgae

All five microalgae species tested showedantibacterial activity against six Vibrio bacteriaBoth in the darkness and in light conditions

Three series of experiments:

1. bacterial communities in cultures of Chlorella minutissima

2. Effect of five microalgae species on Vibrio bacteriaVibrio bacteria

3. Bacterial communities in four species of microalgaeg

9 9

Rhodomonas sp.Phaeodactylum tricornutum

678

678

Log m

L-1

Log m

L-1

50 5 10 15

50 5 10 15

Time (days)Time (days)

9Isochrysis galbana

9Chlorella minutissima

6789

6789

Log m

L-1

Log m

L-156

0 5 10 15Time (days)

56

0 5 10 15Time (days)Time (days) Time (days)

Microalgae cells (X) and total CFU grown in MA (●).

- incubate7 days at 20°C

PROTOCOL

- take 50 ml- shake vigorously

in obscurity - plate in MA dilutions 103, 104, 105

- plate in TCBS dilutions 101- count CFU

Microalgae culture

- incubate1 day at 20°Cin obscurity

- count CFU

in the rest of the volume, measure:- pH- Oxygen

Chlorophyll fluorescence (PEA)

In Flask or bags

- keep bacterial biomass for DNA

- centrifuge 3.200 x g, 10 min

- Chlorophyll fluorescence (PEA)- Absorbance 750 nm- Microalgae cell counts (Malassez cell) - purify the

isolates by 3 passes

biomass for DNA extraction at - 20ºC

- keep the isolates in cryotubes at -80ºC in TSA-li l

- resuspend the pellet in 1ml-centrifuge 10.000 x g, 10 min

- centrifuge the supernatant at 10.000 x g, 10 min-resuspend the pellet in 1ml glicerol- keep the

microalgae pellet at -20ºC

p-centrifuge 10.000 x g, 10 min- keep the bacteria pellet at -20ºC

Algae Water Isolates

DNA extraction

DGGE

Gel analysis and band identification

DNA extraction

José Pintado HCMR, Aprril 2009

Phaeodactylum tricornutum

01 02 03time (days)

Algae Seawatertime (days)

Ph0

Ph0

Ph0

Mk 0 2 6 10 15 Mk 0 2 6 15 Mk

30%

turan

t

1 •

Dena

t

2 •

3 •

4 •

9 •

11 •

5 •

6 •

7 •

8 • 10 •

60%

DGGE profiles of the bacterial communities present in the algae and in theseawater, in a culture of Phaeodactylum tricornutum

time (days)

Rhodomonas sp.Algae Seawater

time (days) 01 02 03

Isolates

04 h05

Mk 0 2 6 10 15 Mk 0 2 6 10 15 Mk Rh Rh Rh Rh Rh

30%

turan

tDe

nat

1 •1

2 •3 •4 •

7 •8 •

9 •

11 • 13 •

60%

5 •6 • 10 •

12 •

DGGE profiles of the bacterial communities present in the algae and in theseawater, in a culture of Rhodomonas sp.

Bag 2t7

Chlorella minutissimaDGGE 03-09-09Gel A

Bag 1 t7

Isolates t6

hl01

h02

hl03

hl04

hl05

Flaskt6

Mk A W A W MkMk A W Mk

30%

Ch Cl Ch Ch Ch

11 •

Mk A W A W Mk

1 •2 •

3 •4 •5 • 6 •

nt

15 • 16 •

17 •

12 •

14 • 1 • 13 •

7 •8 •

9 •

10 •

Dena

turan17 •

21 • 23 •

14 •

18 • 19 •

2 •3 •

4 •5 •6 •

11 •10

12 •13 •

14 •

22 • 15 •

16 •

17 • 18

7 •

9 •8 •

10 •

DGGE profiles of the bacterial communities present in the algae (A) and in the seawater (W), in aculture of Chlorella minutissima in a preculture in Flasks (6 days) in sterile seawater and in a scale

60%

culture of Chlorella minutissima in a preculture in Flasks (6 days) in sterile seawater and in a scale-up culture in bags with non sterile seawater.

so01

so02

so03

IsolatesDGGE 03-09-09Gel B

Isochrysis galbanaAlgae Seawater

30%

Is Is IsMk 0 2 6 10 15 Mk 0 2 6 10 15 Mk

1 • 13 •

turan

t

2 •

3 •

10 •11 •

Dena

t34 •

5 •

6 •

712 •

14 •

15 •

60%

7 •

8 •

9 •

60%DGGE profiles of the bacterial communities present in the algae and in the seawater, in a culture ofIsochrysis galbana at different times, and the predominant isolates from Marine Agar plates at t6.Numbered bands were excised for re-amplification and sequencing. Mk: marker.

Conclusions

1 The DGGE pattern was different for each microalgae species1. The DGGE pattern was different for each microalgae species

2. No changes were observed throughout the trials for each algae

3. In three out of four cases culturable strains were representative for the3. In three out of four cases culturable strains were representative for the

bacteria present in the cultures

Thank you for your attention