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Bioproduction of bioactive compounds : Screening of bioproduction conditions of free-

living microalgae and lichen symbionts

Villa-Carvajal, M.1, Catalá M.2, Barreno, E.3, Tornero Martos, A.J.1

mvilla@ainia.es

1 ainia Centro Tecnológico. Engineering and Processes Department.

Paterna (Valencia) Spain.2 Área de Biodiversidad y Conservación. Universidad Rey Juan Carlos Móstoles (Madrid) Spain.3 Facultad de Ciencias Biológicas. Dpto. Botánica y ICBIBE (Instituto Cabanilles de Biodiversidad y Biología Evolutiva)

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INTRO Bioproduction 04

Materials and methods 08

Results 12

Conclusion 20

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Bioproduction of bioactive compounds-Integrative concept applied and industrial biotechnology-Introduction and objetive of this work

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> TITLE: Bioproduction of bioactive compounds: Screening ofbioproduction conditions of free-living microalgae and lichensymbionts

Why two kinds of “microalgae”?-Free living microalgae: Chlorella and Spirulina sp

Green algae, or Chlorophyta, constitute a huge and extremely diverse phylum of eukaryotic organisms. These eukaryotes must not be confounded with prokaryotic cyanobacteria, also known as blue-green algae. Microalgae biotechnology has been developed for different commercial applications. As photosynthetic organisms, microalgae contain pigments as chlorophyll that can be used for food and cosmetic purposes (Spolaore et al., 2006).

Some species of these genera of microalgae are included in the list of ingredients and food additives in Europe. Application in food industry

These species are described in references such species that contain or produce bioactive compounds that can modulate the immune system in humans

However, microalgae bioproduction represent a unique opportunity to obtain novel or known metabolites at low cost.

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> TITLE: Bioproduction of bioactive compounds: Screeningof bioproduction conditions of free-living microalgae andlichen symbionts

- Microalgae lichen symbiontsAt present, the industrial biotechnological potential using free-living aquatic algae as Chlorella, Chlamydomonas and Dunaliella, sp. (Cadoret et al., 2012) has been developed but the potential of using algae associated with lichens has never been studied.

Asterochloris erici (formerly known as Trebouxia erici Ahmadjian, 1960) is a phycobiont isolated from the northamerican endemic lichen Cladonia cristatella belonging to the class Trebouxiophyceae(Chlorophyta) that has been molecular and phylogenetically well described by Skaloud and Peksa (2010).

Lichens are individualized symbiotic composites (holobionts) of photobionts (oxygenic photoautotrophs, such as cyanobacteria or green algae) that are morphologically and metabolically integrated with

mycobionts (heterotrophic fungi) (Margulis & Barreno 2003).

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OBJECTIVE OF THIS WORK

> TITLE: Bioproduction of bioactive compounds: Screeningof bioproduction conditions of free-living microalgae andlichen symbionts

The possible use of this kind of microalgae for biotechnological purposes requires the development of a

screening of bioprocess conditions studies in order to demonstrate the bioactivity of the compounds and the

technological aptitude that make industrially viable processes.

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Materials and methods

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The bioproductive processes integrateintegrate a set of techniques that use living organisms to make or modify products.

The R & D in bioproduction performing at ainia aims the development and optimization of these processes, through the proper use of physiological and technological methods and always guiding the process from the beginning to find the largest industrial viability.

Bioproduction: Integrative concept of applied andindustrial biotechnology

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BIOPRODUCTION STEPS

HOW?

General skeme of Bioproduction steps

UPSTREAM

BIOPROCESS

DOWNSTREAM

• Culture medium : Synthetic or lab medium and Low cost medium

based on raw agrofood material as vegetable effluents, molasses,

etc.

• Bioprocess Inoculum: microorganisms. Screening and Selection

• Bioproduction of bioactive compounds

• Optimization of Process control parameters (pH, stirring,

Temperature, etc.)

• Recovery of bioactive compounds

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Development of bioproduction process for high value products FROM

MICROALGAE

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•Pretreatments: inoculate and growth media

• Mixothrophystudies (light/obscurity cycles)

• Heterotrophy studies (obscurity)

• on line control (pH, gases, T, mixing, etc.)

Bioproduction monitoring and scale-upControlled conditions bioproduction

Microalgae species selection for high value production

•Preparation of inoculum culture

•species adaptation to Synthetic culture mediums

•species adaptation to low cost culture mediums(molasses)

•Selection based on high value compoundsproduction and its aptitude as industrial microorganism

•Culture monitoring using direct methods (turbidity, optical density) and indirect methods (microorganism kinetics correlation with pigment contents measured by spectrophotometer)

•Microalgae growth stoichiometry

•Energy and mass Balances

•Enthalpic Balances

•Microalgae kinetic

•Modelling and scale-up

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RESULTS

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Free living microalgae: Chlorella and Spirulina

>RESULTS I

1. Species adaptation to low cost culture media

(synthetic glucose & molasses): MIXOTROPHY

2. Cell homogenate treatment influence in bioactivity

3. Downstream: the key of the bioprocess selection based on high value compounds production

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Free living microalgae: Chlorella and Spirulina sp.>RESULTS I

Table 1. Species adaptation to mixotrophy and cell homogenate treatment and itsinfluence in bioproduction of bioactive compounds ( Inmune response modulators NfKb)

---253-SP-A-NA-0,5-SF

+++++252-SP-A-NA-0,5-US

+++++251-SP-A-NA-0,5-CO

+++++250-SP-A-NA-0,5-ST

SP: Spirulinaplatensis (LB)

+++++++++249-VS-A-NA-0,0-US

+++++++++248-VS-A-13M-0,0-US

+++++++++247-VS-A-10G-0,0-US

+++++++++246-VS-A-NA-0,0-CO

+++++++++245-VS-A-13M-0,0-CO

+++++++++244-VS-A-10G-0,0-CO

+++++++++243-VS-A-NA-0,0-ST

+++++++++242-VS-A-13M-0,0-ST

+++++++++241-VS-A-10G-0,0-ST

VS: Chlorella vulgaris

MIXOTROPHY -Synthetic

glucose-Molasses

+++176-ZO-A-NA-0,5-SF

+++++++161-ZO-A-NA-0,5-US

--+146-ZO-A-NA-0,5-CO

+++131-ZO-A-NA-0,5-ST

ZO: Chlorellazofingiensis

++++++116-PY-A-NA-0,5-SF

+++++101-PY-A-NA-0,5-US

++++++86-PY-A-NA-0,5-CO

--+71-PY-A-NA-0,5-STPY:

Chlorellapyrenoidosa

+++++++++56-VS-A-NA-0,5-SF

+++++++++41-VS-A-NA-0,5-US

+++++++++26-VS-A-NA-0,5-CO

+++++++++11-VS-A-NA-0,5-ST

VS: Chlorellavulgaris

2%1%0.1%

ConcentrationSample Code

Microalgaesp.

---253-SP-A-NA-0,5-SF

+++++252-SP-A-NA-0,5-US

+++++251-SP-A-NA-0,5-CO

+++++250-SP-A-NA-0,5-ST

SP: Spirulinaplatensis (LB)

+++++++++249-VS-A-NA-0,0-US

+++++++++248-VS-A-13M-0,0-US

+++++++++247-VS-A-10G-0,0-US

+++++++++246-VS-A-NA-0,0-CO

+++++++++245-VS-A-13M-0,0-CO

+++++++++244-VS-A-10G-0,0-CO

+++++++++243-VS-A-NA-0,0-ST

+++++++++242-VS-A-13M-0,0-ST

+++++++++241-VS-A-10G-0,0-ST

VS: Chlorella vulgaris

MIXOTROPHY -Synthetic

glucose-Molasses

+++176-ZO-A-NA-0,5-SF

+++++++161-ZO-A-NA-0,5-US

--+146-ZO-A-NA-0,5-CO

+++131-ZO-A-NA-0,5-ST

ZO: Chlorellazofingiensis

++++++116-PY-A-NA-0,5-SF

+++++101-PY-A-NA-0,5-US

++++++86-PY-A-NA-0,5-CO

--+71-PY-A-NA-0,5-STPY:

Chlorellapyrenoidosa

+++++++++56-VS-A-NA-0,5-SF

+++++++++41-VS-A-NA-0,5-US

+++++++++26-VS-A-NA-0,5-CO

+++++++++11-VS-A-NA-0,5-ST

VS: Chlorellavulgaris

2%1%0.1%

ConcentrationSample Code

Microalgaesp.

Bioactivity of onespecie (C. vulgaris) in

all concentration...

And treatments?

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Free living microalgae: Chlorella and Spirulina sp.>RESULTS I

Figure 1. Cell homogenate treatment in Chlorella vulgaris and its influence in bioproduction of bioactive compounds ( Inmune response modulators)

C h lo re lla vu lg a r is

0 ,0 0

2 0 ,0 0

4 0 ,0 0

6 0 ,0 0

8 0 ,0 0

1 0 0 ,0 0

1 2 0 ,0 0

1 4 0 ,0 0

0 ,1 1 2

C once ntra t ion (% )

NFkb

act

ivit

y (

ST: S in T ra tam iento

C O : C ho que O sm o tico

U S: U ltra So nido s

Bioactivityincreases withUltrasounds

treatment

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Free living microalgae: Chlorella and Spirulina sp.>RESULTS I

Figure 2. Spirulina platensis downstream represents an industrial handicap: flocculating culture. The recovery is very difficult.flocculating culture. The recovery is very difficult.

3. Downstream: the key of the bioprocess selection based onhigh value compounds production

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Asterochloris ericii photobiont

>RESULTS I I

1. Strain adaptation to liquid and synthetic culture mediums

2. Traditional mixotrophy conditions vs photoautotrophic

3. Massive culture cells: bioproduction system

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Asterochloris ericii photobiont>RESULTS I I

Figure 4. Asterochloris erici growingin solid and liquid culture medium

Figure 5. Asterochloris erici cell lifecycle in liquid medium

Terrestrial and symbioticmicroalgae was adaptedto liquid medium

Cell life cycle may be observed whenmicroalgae grows in liquidmedium …

But…. cell aggregatesappeared and there was a problemproblem to monitor the growth of microalgae and control processing time

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Asterochloris ericii photobiont>RESULTS I I

Figure 6. Asterochloris erici scale-up to200L in FBR

Finally….autotrophic high light conditions make a fact the mass cultivation in liquid medium of this photobiont

cell aggregates dissappeared !!!

and the process was carried out at 200L scale

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Conclusion

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> GENERAL CONCLUSION

The authors are grateful to:

The Generalitat Valenciana and IVACE (Instituto Valenciano de Competitividad empresarial for its support through the project

“INNOVACIÓN EN PROCESOS BIOPRODUCTIVOS PARA LA OBTENCIÓN DE COMPUESTOS DE APLICACIÓN INDUSTRIAL” -INPROBIO

Bioproduction technologies open the door to obtain high-value compounds

from free-living microalgae and lichen symbionts.

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foto

> Ainia Bioproduction web

http://bioproduction.ainia.es/

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Thank you very much for yourattention

Mercedes Villa Carvajal

672 48 10 26mvilla@ainia.es

@Mercedesvillacwww.ainia.es