Process Development for the Production of Microbial Biosurfactants

Transcription

1 Te Industrielle otechnologie mit nachwachsenden Rohstoffen FNR-Symposium, Frankfurt am Main, 11-12th of June 2008 Process Development for the Production of Microbial osurfactants Prof. Dr. Christoph Syldatk University of Karlsruhe (TH) Institute of Engineering in Life Sciences Section II: Technical ology D Karlsruhe, FRG 1

2 Te Outline - The Institute of Engineering in Life Sciences and the Chair of Technical ology at the University of Karlsruhe (TH) - A Short Introduction to Microbial osurfactants - Production of Bacterial Rhamnolipids - Summary and Future Outlook 2

3 Te The new Institute of Engineering in Life Sciences of the University of Karlsruhe (TH) Section I: Food Technology (Head: Prof. Dr.-Ing. Heike Schuchmann) Section II: Technical ology (Head: Prof. Dr. Christoph Syldatk) Section III: oprocess Engineering (Head: Prof. Dr. Clemens Posten) Section IV: oproduct separation (Head: Prof. Dr.-Ing. Juergen Hubbuch) 3

4 Te What is Technical ology? Technical ology deals with the technical application of biological systems or parts of them for the synthesis or the conversion of compounds. The application of biological systems includes bacteria, yeasts, fungi and algae ( Microbial otechnology ) and animal cell cultures ( Cell Culture Techniques ), enzymes, their downstream processing and technical application ( Enzyme Technology ). 4

5 Te Microbial osurfactants for an industrial use? 5

6 Te Microbial biosurfactants Microbial biosurfactants are highly surface and interfacial active compounds consisting of a hydrophobic and a hydrophilic molecule moiety, e.g. a sugar and a fatty acid. They are ecologically well acceptable, biodegradable and many of them are showing interesting biological properties. Most of them are produced extracellularily by microorganisms when growing on water unsoluble substrates to enable their emulsifcation and their uptake into the cell. 6

7 Te Microbial biosurfactants in the 1980ies - Investigations mainly on the use biosurfactants in tertiary oil recovery and bioredimiation - Mainly use of hydrocarbons as substrates for production - Basic research on microorganisms able to produce biosurfactants, chemical structures, surface and interfacial active properties Conclusion: - Highly interesting class of compounds, but too expensive for an industrial use in comparison with chemical surfactants. 7

8 Te The cost limiting factors for the production of microbial biosurfactant in the 1980ies - Substrates too expensive (e.g. pure hydrocarbons) - Product concentrations too low - often caused by substrate and/or product inhibitions - Microbial strains often pathogenic or difficult to handle in larger scale (foam and oxygen transfer problems) - Product mixtures instead of single products requiring high costs for downstream processing and purification 8

9 Te Microbial biosurfactants today - Recent interest for a wider use as detergents and in food, cosmetic and pharmaceutical applications - Of interest as starting material to produce unusual or valuable sugars and fatty acids for nutrition and pharmaceutical applications - Use of renewable resources or organic waste materials as substrates of interest - Availability of commercial enzymes for their modification or treatment to obtain pure products without expensive downstream processing steps - Computer aided process development and metabolic pathway engineering for overproduction possible 9

10 Te Microbial Rhamnolipids 10

11 Te Rhamnolipids are a group of anionic microbial glycolipids produced by Pseudomonas aeruginosa. Four Main Structures are known: Rhamnolipid RL1 Rhamnolipid RL2 Rhamnolipid RL3 Rhamnolipid RL4 11

12 Te History of Rhamnolipid Production Rhamnolipids were first described by Jarvis et al. in They are produced extracellullarily by Pseudomonas aeruginosa especially when grown on hydrophobic C-sources. Rhamnolipids are able to form highly stable emulsions of hydrophobic compounds in aqueous solutions showing low interfacial tensions of <1mN/m and critical micelle concentrations of CMC=10 mg/l-200 mg/l. Rhamnolipids are essential for growth of Pseudomonas aeruginosa on hydrophobic C-sources, but concentrations of <100 mg/l are already sufficient to enable growth. An overproduction of rhamnolipids is only observed under growth limiting conditions or when resting cell conditions are used. 12

13 Te Possible Applications of Rhamnolipids Rhamnolipids: - bioremediation - soil washing or flushing - cosmetic and health care industry - biological control of plant pathogenic fungi - antiviral agents - starting material for L-(+)-Rhamnose. L-(+)-Rhamnose: - chiral compound for the production of pharmaceuticals, plant protection agents, taste and flavour compounds - inducer for recombinant protein synthesis in E. coli - only accessible with difficulty via chemical methods. 13

14 Te Current State of Rhamnolipid Production Depending on the strain, on the process design and on the substrate rhamnolipid concentrations of g/l can be produced with estimated prices of 5-20 US$/kg (Review: Lang and Wullbrandt, 1999). The following processes have been described in literature: - Batch-cultivations under N-limitation using hydrocarbons, plant oils or glycerol as substrates - Fed-batch-cultivations under growth limiting conditions using glycerol, plant oils or ethanol as substrates - Continuous cultivations under Fe-limtations using hydrocarbons as substrates 14

15 Te Current State of Rhamnolipid Production Depending on the strain, on the process design and on the substrate rhamnolipid concentrations of g/l can be produced with estimated prices of 5-20 US$/kg (Review: Lang and Wullbrandt, 1999). The following processes have been described in literature: Only crude rhamnolipid mixtures are currently available on the market at prices, which inhibit a wide range industrial application. Pure rhamnolipids - Batch-cultivations under N-limitation using hydrocarbons, plant oils or glycerol as substrates - Fed-batch-cultivations under growth limiting conditions using glycerol, plant oils areor not ethanol available. as substrates - Continuous cultivations under Fe-limtations using hydrocarbons as substrates - Resting cell processes with free or Alginate-immobilized cells using glycerol as substrate. 15

16 Te Strategies to Enhance Rhamnolipid Production - Avoiding high substrate costs by using renewable resources and waste substrates for rhamnolipid production - Avoiding inhibition of rhamnolipid formation at too high substrate- or biosurfactant concentrations by the use of fed batch processes - Avoiding the use of chemical defoamers by using mechanical foam disruption systems - Overcoming low substrate availabilities of the hydrophobic substrates by the use of integrated membrane for substrate feeding and emulsification - Avoiding foam formation, flotation of the cells and oxygen transfer problems by the use of integrated membrane systems for gasing and degasing of the bioreactor - Avoiding problems with L-2-conditions by screening for non-pathogenic production strains - Using enzymes to produce a single rhamnolipid product, L-(+)-rhamnose and β-hydroxydecanoic acid 16

17 Te The FNR-Rhamnolipid-Network 17

18 Te Forschungsgruppen FAL HO-Pflanzenöle raffiniert/ unraffiniert KD-Pharma Rest-Fettsäuren Glycerinabfälle Substrate FZKA Immobilisierung Schaumfraktionierung Substratauswahl Te KA Substrat/Stamm- Screening Prozessführung FTIR Analytik Stämme Uni Düsseldorf Stammerstellung Stammbereitstellung UPT Saarbrücken Membranentwicklung Prozessintegration Gasaustausch/ISPR Abiotischer Teil bis 12/07 Bruker FTIR Support Feedback Sartorius oprozesstechnik Support Support Nanovation Substrate RL Proben Membranmodule Ecover Test der Substrate Pilotplant ab 2007 ologischer Abbau Henkel Anwendungstests Substrate Anwender Hardware

19 Te Production of Rhamnolipids from renewable resources and waste substrates 19

20 Te FTIR analytics of Rhamnolipids in cell free supernatants 20

21 Te FTIR analytics of Rhamnolipids in cell free supernatants 21

22 Te FTIR analytics of Rhamnolipids in cell free supernatants Rhamnolipid 1 cross validation Rhamnolipid total cross validation 10 8 rhamnolipid 1 cross validation rank = 5 R 2 = RMSECV = RL tot cross validation rank = 5 R 2 = RMSECV = 1.83 predicted [g/l] predicted [g/l] true [g/l] true [g/l] 22

23 Te Formation of rhamnolipids from plant oils and glycerol 23

24 Te Substrate conversion to rhamnolipids Plant oil droplets Glycerol + Oleic acid Rhamnolipids EXTRACELLULAR UPTAKE EXCRETION LIPASE Low substrate solubilities! ß-oxidation of oleic acid and de novo synthesis of Synthesis ß-hydroxydecanoyl- Synthesis and excretion of a lipase ß-hydroxydecanoyl- S-CoA starting from Acetyl-S-CoA Gluconeogenesis starting from glycerol dtdp-rhamnose PSEUDOMONAS AERUGINOSA 24

25 Te Strategies to overcome low substrate solubilities - Overcoming low substrate availabilities of the hydrophobic substrates by the use of integrated membrane for substrate feeding and emulsification [cooperation with UPT, Saarbrücken (Prof. H. Chmiel)] Special feeding membrane or a Microsparger (UPT Saarbrücken) 25

26 Te Plant oil droplets induce rhamnolipid formation. Induction of RL synthesis by the hydrophobic plant oil or by hydrocarbons necessary Adsorption of RL by the plant oil as protection against product inhibition (the substrate acts as a reservoir) Emulsification of the hydrophobic substrate necessary for substrate uptake 26

27 Foam formation and defoaming agents cause problems during rhamnolipid production 40 RL Rhamnolipid g/l Te 30 Antischaumzugabe 1% (v/v) Pv= 0,33-0,39 g/lh Prozesszeit in h Defoaming agents seem to cause a limitation of rhamnolipid formation(?). 27

28 Te Advantages of Mechanical Foam Disruption - No use of defoaming agents necessary - No toxic effects on the microorganisms - No negative influence on oxygen transfer rate - Less problems in down stream processing - Lower costs 28

29 Te Scale down for experiments on substrate screening in a parallel Sixfors bioreactor system Mechanical foam disrutors for Sixfors Parallel oreactor System Sixfors oreactor System for Substrate Screening Experiments 29

30 Result: A Fed Batch Process for Rhamnolipid Production BTM Feed Öl RL 40,00 120,00 35,00 100,00 80,00 25,00 20,00 60,00 15,00 40,00 Öl [g/l]; RL [g/l] 30,00 BTM[g/L] Te 10,00 20,00 5,00 0,00 0, Prozesszeit [h] 30

31 Te Screening for different substrates for the formation of rhamnolipids by Pseudomonas aeruginosa Rape Seed Oil Sunflower Oil Corn Oil Fish Oil Fractions HOS 90+ Sunflower Oil HOS 85* Sunflower Oil Refined and Non-Refined Oils Different yields, but comparable productivities No influence on the product composition 31

32 Te Comparison of different Pseudomonas aeruginosa production strains DSM 2874 and DSM

33 Te Suboptimal conditions are leading to the formation of exopolysaccharides and highly viscous media. Alterations in medium composition and oxygen supply can lead to the production of exopolysaccharides instead of rhamnolipids. 33

34 Te Screening for Alternative Production Strains 34

35 Te Screening for Alternative Production Strains - Pseudomonas aeruginosa is an L-2-strain! - Preselection of alternative strains from the literature - Colorimetric CTAB-agar plate screening system allows fast detection of biosurfactant producing strains - Optimisation of biosurfactant production in shake flask and bioreactor experiments - Product isolation, characterisation and structure elucidation - Scale up experiments to 30-litre bioreactor 35

36 Te Assay for alternative production strains Burkholderia glumae Burkholderia plantarii Pseudomonas chlororaphis Pseudomonas cruciviae Pseudomonas oleovorans Pseudomonas putida Tetragenococcus koreensis CTAB = CetyltrimethylAmmoniumbromid zum Nachweis anionischer otenside 36

37 Te A Rhamnolipid with alterated structure and different HLB-value from the strain Burkholderia plantarii -Q1: 5 MCA scans from Sample 1 (TuneSampleID) of Q1-B-plantarii ppm-L3-IS-neg.wiff... Max. 2.2e7 cps e7 2.0e7 1.8e7 1.6e7 1.4e7 1.2e7 1.0e7 8.0e6 6.0e6 4.0e e m/z, amu Recent activities: Structure elucidation and characterisation concerning surface and Interfacial active properties (cooperation with FZK (M. Heyd) and the Lang group, TU Braunschweig).

38 Te Enzymatic conversion to obtain MonoRhamnolipid, pure L-(+)-Rhamnose and β-hydroxydecanoic acid 38

39 Te Enzymatic conversion to obtain Mono-Rhamnolipid, pure L-(+)-Rhamnose and β-hydroxydecanoic acid HO HO O O O OH O HO OH O Naringinase COOH OH O OH HO O OH mono-rl di-rl O O O O OH COOH OH OH L-rhamnose NARINGINASE: Conversion of Di-Rhamnolipid Enzyme from Penicillium decumbens -L-Rhamnosidase - ß-Glucosidase Enzyme complex commercially available Glycoprotein of 90 kda 39

40 Enzymatic conversion to obtain Mono-Rhamnolipid, pure L-(+)-Rhamnose and β-hydroxydecanoic acid HO HO HO O OH O O OH RL Gesamtmolanteil ( / ) Te O O COOH O OH Step 1 OH HO O OH O O O COOH O COOH Step 2 di-hda mono-rl di-rl O The consecutive conversion (step 2) is 55 times slower than step Time(min) (min) Zeit 40

41 Te Summary - Microbial biosurfactants and especially rhamnolipids are of high interest for industrial applications. Renewable resources and waste substrates are well suitable substrates for their production. - FTIR is a suitable fast and simple detection method for the quantitative analysis of rhamnolipids. - The product composition of rhamnolipids is not dependent on the nature of the substrate. - The problems of substrate inhibition and foam formation can be overcome by optimisation in process engineering. - The glycosidic enzyme Naringinase can be used to produce pure RL 1, L-(+)-rhamnose and β-hydroxydecanoic acid. 41

42 Te Future Outlook the Opti-R -Project - Systems biology and prediction of metabolic engineering experiments [cooperation with TU Braunschweig] - Production strain development by genetic engineering methods [cooperation with University of Düsseldorf] - Process development with alternative production strains [University of Karlsruhe] - Development of methods for a solvent free downstream processing of rhamnolipids [University of Karlsruhe] 42

43 Te Who is doing the work in Karlsruhe? Dr.-Ing. Rudolf Hausmann, Assistant Professor Dipl.-ol. (t.o.) Frank Leitermann, Ph.D. student Dipl.-Ing. Ivana Maria Magario, Ph.D. student Dipl.-ol. (t.o.) Vanessa Walter, Ph.D. student Master Craftman Siegfried Almstedt 43

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