Interaction of plant growth-promoting bacteria and microalgae: from basic studies of plant–bacteria

interaction to potential biotechnological applications

Luz de Bashan, Edgar Amavizca, Juan Pablo Hernandez, Blanca Lopez, Oskar

Palacios, Yoav Bashan Environmental Microbiology Group,

Northwestern Center for Biological Research (CIBNOR) Mexico

and Bashan Foundation, The Bashan Institute of

Science (USA)

Our proposal: Immobilization of the green microalga Chlorella with the PGPB Azospirillum brasilense

Why this association? - Chlorella is consider an

unicellular plant - Azospirillum is a unespecific

PGPB - It is possible that Azospirillum

would affect Chlorella, in the way it does with higher plants

Interaction, forcing a physical contact, inside alginate beads

microalgae

cavities

de-Bashan et al, 2011, Journal of Phycology 47: 1350-1359

Movement of Azospirillum towards Chlorella inside alginate beads

Azospirillum in cavity

Alginate bead

1 µ

Lebsky et al. 2001. Canadian Journal of Microbiology 47:1-8

Chlorella

Days of incubation

Dis

tanc

e be

twee

n co

loni

es o

f C. v

ulga

ris

and

A. b

rasi

lens

e (µ

m)

0

1

2

3

4

5

6

de-Bashan et al. 2011, Journal of Phycology 47: 1350-1359

1 3 7 10

Association of Chlorella and Azospirillum immobilized in alginate beads – Fluorescence in situ Hybridization (FISH).

Azospirillum

Chlorella Chlorella

Azospirillum

de-Bashan et al, 2011, Journal of Phycology 47: 1350-1359

Azospirillum

Chlorella

Attachment of Azospirillum to the microalgae - SEM

de-Bashan et al, 2011, Journal of Phycology 47: 1350-1359

Model of interaction

de-Bashan et al. 2012. Applied Soil Ecology 61: 171-189

Effect of Azospirillum brasilense on Chlorella vulgaris

Gonzalez and Bashan 2000 Applied and Environmental Microbiology 66: 1527-1531 de-Bashan et al. 2004 Water Research 38: 466-474 Choix et al. 2012. , Enzyme and Microbial Technology 51: 294-299

Growth

00.5

11.5

2

2.53

3.54

1 2 3 4 5 6

Total carbohydrates N

o. c

ells

106

. be

ad-1

de-Bashan et al. 2002. Can. J. Microbiol. 48, 514-521 Leyva et al. 2015. Annals of Microbiology 65: 339-349 Leyva et al. 2014. Naturwissenschaften 101:819–83

Total content of lipids and fatty acid in Chlorella immobilized with A. brasilense

Fatty acid Increased (%)

Palmitic (16:0) 172

Palmitoleic (16:1) 443

Heptadecenoic (17:1 ω7) 156

Stearic (18:0) 145

Oleic (18: 1 ω 9) 268

Linoleic (18:2 ω 6) 130

Linolenic (18: 3 ω 3) 402

Arachidic (20:0) 100 0

50

100

150

200

250

300

350

400

C. sorokin iana C. vu lgaris

Tota

l lip

ids

(ug

/g D

W)

0

100

200

300

400

500

600

700

800ug

/ g

cells

Chlorophyll a Chlorophyll b Lutein Violoxanthin

Thiamine

Effect of Azospirillum brasilense on Chlorella vulgaris

Pigments

de-Bashan et al. 2004 Water Research 38: 466-47 Palacios et al. Journal of Applied Phycology, submitted

0

0.3

0.6

0.9

1.2

1.5

Chlorella alone Jointly immobilized

AC

Cas

e ac

tivi

ty .

mL

-1

0

5

10

15

20

0 24 48 72 96 120 144U

mg

prot

ein

-1

AGPase

Effect on enzymatic activities - in Chlorella

AcetylCoA Carboxylase

Hours of incubation

Choix et al. 2014. Journal of Biotechnology 177: 22-34 Leyva et al. 2015. Annals of Microbiology 65: 339-349

0

1

2

3

4

5

6

7

8

Act

. cel

l x 1

0-6

Glutamine synthetase

Glutamate dehydrogenase

de-Bashan et al. 2008 Journal of Phycology 44: 1188–1196

Effect on enzymatic activities – in Chlorella

Effect on enzymatic activities – in Azospirillum

ipd carboxilasa

Palacios et al. Research in Microbiology, submitted

No.

cel

ls X

106

/ml

0.2

0.4

0.6

0.8

1.0

1.2

1.4C. vulgarisC. vulgaris + A. brasilense Sp245 C. vulgaris + A. brasilense FAJ0009

2 3

Aa

Ab

Bb

Bc

Effect of IAA produced by Azospirillum on the growth and intracellular accumulation of N and P in Chlorella

de-Bashan et al. 2008 Journal of Phycology 44, 938-947, Meza et al. 2015, Research in Microbiology, 166: 72-83 Meza et al. 2015 Research in Microbiology, in press

0

0.5

1

1.5

2

mg

NH 4

+

Cv + FAJ0009 Cv + SpM Cv + Sp245 Cv + Sp6

Pg

P ce

ll-1

Tryptophan (Chlorella) – IAA –(Azospirillum)

Palacios et al. Research in Microbiology (submitted)

Is there a transfer of elements between the partners?

CIBNOR (Drs. Bashan and de-Bashan); NASA Ames Research Center (Dr. Brad Bebout)- Lawrence Livermore National

Laboratory (Dr. Xavier Mayali)

Analysis of cell-to-cell transfer, by NanoSIMS

Secondary Ion Mass Spectrometry (SIMS) Microprobe for Isotopic and Trace Element Analysis at High Spatial Resolution

a b

Azospirillum 15N and 13C labeled, incubated with unlabeled Chlorella for 4 days

a b

de-Bashan et al. 2015, PNAS submitted

Unlabeled Azospirillum incubated with 13C labeled Chlorella for 4 days

b a

de-Bashan et al. 2015, PNAS submitted

Conclusion

• The microalgae Chlorella spp. responds to association with Azospirillum in similar way as do higher plants

• The immobilizing system with two organisms is easy to handle, reproducible, returns fast results (on microbial time scale), and is inexpensive.

• This two-organisms immobilizing system is presented as a practical model for basic studies of physiology and molecular biology of plant-microbe interactions

All papers are available as PDF at www.bashanfoundation.org