microbial conversion of agroindustrial wastes

A. Manresa Unitat de Microbiologia Facultat de Farmcia Universitat de Barcelona

 microbial diversity and products process optimization: complex substrates new horizon : integrated process applications

ecoeficiencia global optimisation de un sistema Nuevos horizontes de produccin, en trminos ecolgicos

aumenta la responsabilidad acerca del impacto ambiental de la industrializacin valoracin del impacto consumo de los recurso, de energa, de emisiones, del riesgo potencial para la salud y el entorno

Candida bombicola
Soforolipids

Torulopsis apicola Yeast Pseudozima Candida

Mannosylerythritol lipids

Tsukumarella Oligosaccharide lipids Saccharomyces Kluveromyces

mannanoprotein

Fungi

Geotrichum candidum Ustilagus maydis

Mannosylerythritol lipids Cellobiose-lipids
Lang Current Opinion in Colloid & Interface Science 7:1220, 2002.

Microbial Biosurfactants and Emulsifiers

Molecules with surface activity Low molecular weight ! decrease of surface and interfacial tension High molecular weight !emulsifier agents Produced by bacterias yeast or fungi molecular agregates (miceles) Biodegradables and environmental frienddly

Flavobacterium

Gram positives

Proteobacteria

Bacteria

(Bacteroidetes) Flavobacteria Ornithine-lipids Flavobacterium sp Flavolipids Myroides odoratum

Gram+ low CG Tetragenococcus

Rhamnolipids

Lactobacilus Bacillus

surlactin Iturine A

lipopeptide

Surfactin Polymixin

Glycolipid

Gram+ high GC

Rhodococcus Arthrobacter
Carbohydrate 40% Lipid 27% Protein 29%

HO H
COO

H H O OOC H

OH

OH

Trehalipids
H

H H
OOC

O H H
OOC

OH

COOH

Cellulomonas Renibacterium Nocardioides Mycobacterium

Glycolipid

Microbacterium

Rhamnolipids

Corynebacterium
lipid-mycolate
R: C15, C16, C17

glycerolipids

Esch et al Carbohydrate Research 319 (1999) 112123 Palme et al Biosurfactants, p185 (2120)

Gluconobacter

lipo-ornitine

"
Proteobacteria Alcaligenes
glycolipid

Sphingobacterium Sphingolipids Rhamnolipids Burkholderia

#
Myxococcus Serratia

???
Lipopeptide Serrawetina

%
Proteobacteria Pseudomonas

Rhamnolipids Lipopeptides (masetolides) fatty acids

OH

OH COOH

Acinetobacter Alasan Phospholipids

Emulsan

 microbial diversity and products process optimization: complex substrates new horizon : integrated process applications

Criteria for microbial production

the use of cheap or waste substratres characterization of new products process development

Mercad & Manresa JAOCS 71:61-64(1994)

Central composite design of four factors: responses

20 15 10 5 0 -2 -1 X2 0 1 2 -2 1 X1 RL s (g/L )

carbon/nitrate

20 15 10 5 0 -2
12

RLs (g/L)

1 -1 0 X4 1 2 -2 X3

Nitrate/iron
Bi omasa (g/L )

10 8 6 4 2 -2 -1 X2 -2 1 0 X1 1

Carbon/nitrate 0

12

Biomasa (g/L)

10 8 6 4 2 0 -2 -2

Phosphate/Iron
1 X3

Abalos et al J Chem Technol Biotechnol 77:777 (2002)

Ramnolpidos: Superficies de Respuesta

20 15 10 5 0 -2 -1 X2 0 1 2 -2 1 X1

RL s (g/L )

Carbono (X1) - Nitrato (X2)

( X1; X2; X1 X2; X12; X22; X1 X22 )

2 1

18-20 16-18 14-16 12-14 10-12 8-10 6-8 4-6 2-4

Y1

0 X1 -1 -2 -2 -1 0 X2 1 2

Fosfato (X3) = 1g/L Hierro (X4) = 7,4 mg/L

Ramnolpidos: Superficies de Respuesta

20 RLs (g/L) 15 10 5 0 -2 -1 0 X4 1 2 -2 1 X3

Fosfato (X3) - Hierro (X4)

2 1 18-20 0 X3 -1 -2 -2 -1 0 X4 1 2 16-18 14-16 12-14 10-12

Y1

Carbono (X1) = 50 g/L Nitrato (X2) = 4,64 g/L

Ramnolpidos: Superficies de Respuesta

20 RL s (g/L) 15 10 5 0 -2 -1 0 X4 1 -2 2
Y1

Nitrato (X2) - Hierro (X4)

( X2 X4 )
1 X2

2 1 0 X2 -1 -2 18-20 16-18 14-16 12-14 10-12 8-10 6-8

Fosfato (X3) = 1g/L Carbono (X1) = 50 g/L

-2 -1 0 X4 1 2

Biomasa: Superficies de Respuesta

12 Bi omasa (g/L ) 10 8 6 4 2 0 -2 -1 X2 0 1 2 -2 1 X1 2 1 0 X1 -1 -2 -2 -1 0 X2 1 2 10-12 8-10 Y2 6-8 4-6 2-4 0-2

Carbono (X1) - Nitrato (X2)

Fosfato (X3) = 7 g/L Hierro (X4) = 13,75 mg/L

Biomasa: Superficies de Respuesta

12

Biomasa (g/L)

10 8 6 4 2 0 -2 -1 0 X4
Y2

Fosfato (X3) - Hierro (X4)

1 1 2 -2 X3

2 1 11-12 0 -1 -2 -2 -1 0 X4 1 2 X3 10-11 9-10 8-9

Carbono (X1) = 50 g/L Nitrato (X2) = 9 g/L

Optimization of the culture medias: partial effects Biomass

12 10

Biomasa (g/L)

8 6 4 2 0 -2 -1 0 1 2

Valores codificados
Carbon carbono
nitrato Nitrate fosfato Phosphate

Iron hi erro

Optimization of the culture medias: partial effects Rhmanolipids

20 17 14

RLs (g/L)

11 8 5 2 -2 -1 0 1 2

coded factors
Carbon carbono

nitrato Nitrate

fosfato Phosphate

Iron hierro

Abalos et al J Chem Technol Biotechnol 77:777 (2002)

16 10
Biomasa, RLs (g/L) RLs, Biomasa (g/L)

8 12

10,50 g/L

16,50 g/L

6 5 4 3

10
6

6 4

8 4 2
0 12 24 24 36 48 60 60 72 84 96

4 6 2

2 2 1 0
Tiempo (h) Tiempo (h) Biomasa Ramnolpidos Ramnolpidos Biomasa pH pH

Nitratos nitrato

Abalos et al J Chem Technol Biotechnol 77:777 (2002)

Nitrato (g/L), pH Nitrato (g/L), pH

14

8 7

Rhamnolipid optimization Rhamnolipid optimization

initial medium initial medium

dessigned medium dessigned medium terico terico experimental experimental 16,50 16,50 4,50 4,50 3,60 3,60 33,0 33,0 0,20 0,20

RLs (g/L) RLs (g/L) Biomass (g/L) Biomass (g/L) YP/X (g/g) YP/X (g/g) YP/C (%) YP/C (%) P(g/Lh) P(g/Lh)

3,60 3,60 4,23 4,23 0,85 0,85 18,0 18,0 0,04 0,04

18,6 18,6 5,6 5,6 3,3 3,3 37,3 37,3 0,2 0,2

surface tension of ramnolpidos produced by Pseudomonas aeruginosa from waste substrates

cepa 47T2 AT10 AT10 AT10 47T2 LB1

substrato OOME soja waste soja waste soja waste waste frying oil soapstock

producto nd M8 R2 C10C10 M6 M8 M6

TS (mN/m) 30 26,8 28.8 27,3 32.8 24

TI (mN/m) 5 nd <1 nd 1 1,3

cmc (mg/L) nd 150 106 234 109 120

Mercad et al (1993) Abalos et al (2001) Abalos et al (2001) Abalos et al (2001) Haba et al (2003) Benincasa et al (2004)

Mercad et al. Biores Technol 43:1-6 (1993) Haba et al. Biotechnol Bioeng 81:317-322 (2003)

Abalos et al. Langmuir 17:1367-1371 (2001) Benincasa et al. ANTO 85:1-8 (2004)

propiedades fsico-qumicas ramnolpidos de Pseudomonas aeruginosa

cepa 47T2 AT10 AT10 AT10 BS2 47T2 LB1 J4 PA1 EBN-8 substrato OOME residuo soja residuo soja residuo soja suero leche aceites fritura soapstock petroleo WW glicerol residuos aceites producto nd M8 R2 C10C10 M6 nd M8 M6 M4 cultivo nd TS (mN/m) 30 26,8 28.8 27,3 27 32.8 24 31 30 28,5 1 1,3 nd 2 nd 109 120 50 60 150 TI (mN/m) 5 nd <1 nd cmc (mg/L) nd 150 106 234
Mercad et al (1993) Abalos et al (2001) Abalos et al (2001) Abalos et al (2001) Dubey et al (2001) Haba et al (2003) Benincasa et al (2004) Wei et al (2005) Santa Ana et al (2006) Ali-Raza et al (2007)

Chemical caracterization of rhamnolipids

5 4

16.5 g/L 12.3 g/L

RLs (g/L)

3 2 1 0 0
R1C8:2 R1C10C12:1 R2C10C12:1

4.15 g/L 0.21 g/L

24 48
R1C10C10 R2C10C10 R1C12:2

72

96 Tiempo (h)
R1C10C12 R2C10C12

Haba et al. J Surf Det 6:155-161 (2003)

 microbial diversity and products process optimization: complex substrates new horizon : integrated process applications

Produccin de bioetanol

total C: 8,5

VINASSE
(10L/L ALCOHOL)

N: 0,69 P2O5: 0,004 K2O: 3,3 CaO: 1,53 MgO: 0,58

(g/L) pH: 3,5

ALCOHOL

Produccin de Aceites Vegetales

Extraction Process Crude oil Refining Process

Deguming Process

linoleic acid: 51,4% oleic acid: 4,5% pamitic acid: 16,8% stearic acid: 2,9% linolenic acid: 4,4%

Gums

Centrifugation Alkali

Soapstock

Neutralizing Process

pH: 11-12

id 2% Fatty Ac g/L COD:2.700 m

Wastewater

pH: 2-3

Whashing Process

Waxes

Dewaxing Process

Deodorization Process

Bleaching Process

Refined Vegetable oil

rhamnolipid production in an integrated process

50 40 % ST(mN/ m) 30

% ST
20

50
10 0 0 150 300

pH2 pH4 pH 6 pH8

45 40 600 35 RL (mg /L) 30 (m N/m )

450
ST

750

900

70 60 %ST (mN/m) 50 40
1000

pH10

1% NaCl 1,5%NaCl 2%NaCl 2,5%NaCl

25 20 0 200 400

60030 800

1200

RL (mg/L) 20

10 0 0 200 400 600 800 1000 1200 RL (mg/L)

Benincasa et al IJEE in press (2011)

RLs (%)

RGLA soja

Frying oils

soapstock sunflower

soapstock vinasse

R2C10C10 R1C10C10 R2C10C12:1 R1C10C12:1 R2 C10 C12 R1 C10 C12 R2 C12C12:1 R1C10 C8 R2C10 C8 R1C8:2 R1C12:2 R2C8C12

31,9 25,2 6,7 7,1 12,9 2,8 6,8 6,4 22,8 50,6 6,8

35,2 23,3 6,6 10,2 12,0 10,2 1,3 1,2 41,6 28 -

28,9 23,4 23,3 7,9 11,3 5,5 25 50 -

22 54,4 3,9 3,9 0,6 9,3

Effect of the substrate on composition

oleic acid (C18:1) linoleic acid (C18:2) linolenic acid (C18:3)

RLs (%)

soapstock vinhaa

RGLA soja

aceites fritura

soapstock girasol

R2C10C10 R1C10C10 R2C10C12:1 R1C10C12:1 R2 C10 C12 R1 C10 C12 R2 C12C12:1 R1C10 C8 R2C10 C8 R1C8:2 R1C12:2 R2C8C12

22 54,4 3,9 3,9 0,6 9,3

31,9 25,2 6,7 7,1 12,9 2,8 6,8 6,4 -

35,2 23,3 6,6

50 10,2 40 12,0 % ST(mN/ m)

28,9 23,4 23,3 7,9 11,3 5,5

% ST

10,2 30 - 20
10 0 0 150

1,3 1,2 -

300 450 600 750 900 RL (mg /L)

 microbial diversity and products process optimization: complex substrates new horizon : integrated process applications

propiedades y aplicaciones de los biotensioactivos propiedades y aplicaciones de los biotensioactivos

! estimuladores del crecimiento: solubilizacin susbtratos ! estimuladores del crecimiento: solubilizacin susbtratos ! biomedicina: antimicrobianos, esporicidas, antitumorales ! biomedicina: antimicrobianos, esporicidas, antitumorales ! captador de metales: detoxiciacin, restauracin ! captador de metales: detoxiciacin, restauracin ! aditivos formulaciones: emulsionantes ! aditivos formulaciones: emulsionantes ! nucleadores congelacin: formacin cristales hielo ! nucleadores congelacin: formacin cristales hielo !MEOR: extraccin petrleo, limpieza tanques !MEOR: extraccin petrleo, limpieza tanques ! restauracin de suelos: bio-remediacin ! restauracin de suelos: bio-remediacin

Microorganismo
Alcaligenes faecalis Bordetella bronchiseptica Citrobacter freundii Enterobacter aerogenes Escherichia coli Salmonella thyphimurium Serratia marcescens Bacillus cereus v. mycoide Bacillus subtillis Microccocus luteus Mycobacterium phlei Staphyloccocus aureus Staphyloccocus epidermidis Streptoccocus faecalis Aureobasidium pullulans Aspergillus niger Chaetomium globosum Gliocadium virens Penicillium chrysogenum Penicillium funiculosum Botrytis cinerea Colletotrichum gloeosporoides Rhizoctonia solani Fusarium solani

residuo aceite soapstock soja frtura girasol

32 128 >256 >256 32 128 16 64 64 32 16 128 8 64 32 16 32 16 32 128 18 65 18 65 64 128 64 4 64 128 8 64 16 64 128 32 32 nd >256 >256 64 32 >256 16 170 256 109 75 nd 128 nd 4 >256 16 nd 4 8 128 nd 8 >256 4 64 32 32 64 32 nd nd nd nd nd

actividad antimicrobiana ramnolpidos producidos por Pseudomoas aeruginosa

Binding of DiRhamnolipid to BSA membranes

A: Phosphatidylcoline B: Dirhamnolipid C: Phosphatidilethanolamine

Ortiz et al Int. J Pharm 325:99-107 (2007) Sanchez et al Langmuir 24:6487-6495 (2008)

Comparacin entre los trealosalpidos producidos por R.erythropolis 51T7 y los ramnolpidos producidos por P.aeruginosa AT10
0
Petroli

oil

100

Petroli

oil

100

a/t/p

p/t/a
50 50 50

Threaloselipids

50

100
water Aigua

0 0 50 100
oil Petroli

100 0 50 100
Petroli oil

0
ThL Trh-l

ThL Trh.lpid water Aigua

100

100

50

50

50

50

Rhamnolipids
100
Aigua water 0

0 50 100
RL Rmn

100
Aigua water

0 0 50 100
RL Rmn

Marqus et al. Chem Phys Lipids 158:46-53 (2009)

Casablanca oil biodegradation

CONTROL

AM

AM+Ls

oil

120 100 80 60 % PAH 40 20 0

H2O

RLs

10

Time (days) Abalos et al Biodegradation 15:249-260 (2004)

AM-10 Naftaleno Metilnaftaleno Dimetilnaftaleno Trimetilnaftaleno Tetrametilnaftaleno Fluoreno Metilfluoreno Dimetilfluoreno Trimetilfluoreno Fenantreno/Antraceno Metil Fn/An Dimetil Fn/An Trimetil Fn/An Metil-DBT Dimetil-DBT Trimetil-DBT Metilpireno 11,5 13,8 11 0 0 30 0 0 0 36 0 0 0,3 0,5 0,9 8,5 0

AMRLs-10 94,7 92,2 99,0 95,5 17,0 100 89,9 14,6 44,3 72,8 88,5 56,4 39,0 81,0 47,8 34,6 35,4

AM-20 100 100 100 87 11 100 87 18 9 100 96 48 20 75 40 29 -

12,52%

62,28%

63,75%

Utilizacin de efluentes urbanos para la produccin de tensioactivos

Alga lipognica

residuos municipales digestor anaerbio

CO2
hidrlisis fangos activados
CH4

Lipidos microbianos (SCO)

celulosa

efluente
Productor de tensioactivos

Biomasa celular tensioactivos

ecoeficiency: global optimisation of the production in ecological terms

impact valorization: consume of materia (biomass, water), energy; emissions, health and environmental risk enlargement the life cycle of materials and resources enhance responsability: environmental impact of industrialization

development of new technologies based on renewable resources