Effects of acetic acid, furfural and p-hydroxybenzoic acid on succinic acid fermentation by ...

85 Pages·2014·7.58 MB·English

MASTER'S THESIS
Effects of Acetic Acid, Furfural and P-
hydroxybenzoic Acid on Succinic Acid
Fermentation by Escherichia coli BSS133
Mireille Ginésy
2014
Master of Science in Engineering Technology
Chemical Engineering
Luleå University of Technology
Department of Civil, Environmental and Natural Resources Engineering
Effects of acetic acid, furfural and p-hydroxybenzoic acid
on succinic acid fermentation by Escherichia coli BSS133
MireilleGine´sy
Thursday20th March,2014
1
2
Abstract 3
Abstract
Duringhemicellulosehydrolysis,amandatorysteppriortofermentation,degradationproducts
areformedfromsugarsandligninInthecaseofhardwoodhemicellulose,acetateisalsoreleasedfrom
xylan deacetylation. Those compounds might be inhibitory for both Escherichia coli growth and
the succinic acid production. In the present work, E. coli growth was first tested on both glucose
and xylose in the presence of different inhibitors: sodium acetate [0-12 g/L]; furfural [0-3 g/L]
and4-hydroxybenzoicacid[0-2g/L].Thisinvestigationwasperformedinshakeflasks,usingsmall
size inocula. Afterwards, eleven fermentations in batch mode were carried out in 1-L fermentors,
following a full factorial design with three centred points. The ranges of inhibitors studied were:
aceticacid[0-10g/L],furfural[0-2g/L]andp-hydroxybenzoicacid[0-2g/L].Finally,hemicellulose
was extracted from birch wood, hydrolysed with 2.7% sulfuric acid and used as a substrate for a
fermentation.
The results showed that furfural was the most toxic compound. Besides, its association with
acetic acid was synergistic since no growth occurred when furfural and acetic acid were present
in the media at 2 and 10 g/L, respectively. Nevertheless, E. coli was able to hydrolyse these two
compounds to some extent. Low POH concentrations did not have any significant effect on the
growthandfermentationperformanceof E.coli. Aceticacidathighconcentrationsaffectedboththe
growthandthesuccinicacidproduction. Indeed, lowersuccinicacidconcentrationswereobtained
when there was an initial concentration of 10 g/L of acetic acid in the media. Acetic acid, furfural
andphenolswerefoundinthehydrolysatesatconcentrationof13.3g/L,2.2g/Land1.5g/Lrespec-
tively. AsexpectedfromtheexperimentalmodelE.coliwasthusnotabletogrowonundetoxified
birtchwoodhemicellulosehydrolysateswhenusingabatchprocess.
4 Preface
Preface
Thisthesisworkwasconductedinspring2011atLulea˚ UniversityofTechnology(LTU).
It was financially supported by grants from the Swedish Energy Agency and Swedish
GovernmentalAgencyforInnovationSystem(VINNOVA).
FirstofallIwouldliketothanksProfessorKrisBerglundforallowingmetoperformmy
masterthesisprojectwithinthebiochemicaltechnologygroup. Iwouldalsoliketoexpress
mygratitudetoProfessorUlrikaRova. ThankyouforprovidingmeasubjectthatIcameto
likeverymuch, despiteinitialdoubts. FinallyIwouldliketothanksmysupervisorJonas
Helmerius. Ilearnedalotunderyoursupervision.
Contents
1 Introduction 10
1.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.2 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2 Succinicacid 12
2.1 Succinicacidandindustry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.1.1 Marketsandapplications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.1.2 Chemicalsynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.1.3 Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.2 Succinicacidfermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2.1 Differentsuccinicacidproducers . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2.2 E.coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.2.3 MixedacidfermentationinE.coli . . . . . . . . . . . . . . . . . . . . . . . 18
2.2.4 ProductionofsuccinicacidbyE.coli . . . . . . . . . . . . . . . . . . . . . . 20
3 Utilisationofhardwoodhemicelluloses 21
3.1 Hemicellulosestructure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2 Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.3 Inhibitorsinhydrolysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.3.1 Generationofinhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.3.2 Inhibitorsinhardwoodhemicellulosehydrolysates . . . . . . . . . . . . . 26
3.4 Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.4.1 Inhibitionmechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.4.2 Inhibitioneffects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.5 Overcominginhibitionproblems . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.5.1 Detoxification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.5.2 Processdesign. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.5.3 Strainimprovement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4 Methods 43
4.1 Bacterialstrain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.2 Growthinvestigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.2.1 Culturemedium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.2.2 Inoculumpreparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.2.3 Cultureconditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.3 Fermentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.3.1 Experimentaldesign . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.3.2 Inoculumpreparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.3.3 Reactormediumpreparation . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5
6 CONTENTS
4.3.4 Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.4 Hydrolysates: preparationandfermentation . . . . . . . . . . . . . . . . . . . . . 47
4.4.1 Hemicelluloseextractionandhydrolysis . . . . . . . . . . . . . . . . . . . 47
4.4.2 Mediapreparationandfermentation . . . . . . . . . . . . . . . . . . . . . . 47
4.5 Analyticalprocedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.5.1 Celldensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.5.2 Cellviability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.5.3 Sugars,inhibitorsandsuccinicacidanalysis . . . . . . . . . . . . . . . . . 49
4.5.4 POHconcentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.6 Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.6.1 Specificgrowthrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.6.2 Biomassyield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.6.3 Volumetricproductivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.6.4 Productyield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
5 Results&analysis 51
5.1 Growthinvestigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
5.1.1 Evolutionofthecelldensity . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
5.1.2 Evolutionoftheinhibitorsconcentrations . . . . . . . . . . . . . . . . . . . 52
5.2 FermentationsResults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5.2.1 Duplicates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
5.2.2 Growthphase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
5.2.3 Productionphase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.2.4 Evolutionofinhibitorsconcentrations . . . . . . . . . . . . . . . . . . . . . 58
5.3 Fermentationsanalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5.3.1 PLSmodel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5.3.2 Analyticalresults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.4 Hydrolysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
6 Discussion 67
7 Conclusion 70
8 Futureprospects 71
Listofabbreviations 72
References 84
List of Figures
Figure1. Basicprincipleofabiorefinery . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Figure2. Structureofsuccinicacid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Figure3. Potentialusesofbiobasedsuccinicacid . . . . . . . . . . . . . . . . . . . . . 13
Figure4. Chemicalsynthesisofsuccinicacid . . . . . . . . . . . . . . . . . . . . . . . 14
Figure5. ScanningelectronmicrographofEscherichiacoli . . . . . . . . . . . . . . . . 17
Figure6. HexoseandpentosemetabolisminE.coli. . . . . . . . . . . . . . . . . . . . 18
Figure7. Tricarboxylicacidcycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Figure8. MixedacidfermentationpathwayinE.coli . . . . . . . . . . . . . . . . . . . 19
Figure9. StandingvolumeofdifferenthardwoodspeciesinSweden . . . . . . . . . 21
Figure10. Plantcellwall. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Figure11. Structureofglucuronoxylanfromhardwood . . . . . . . . . . . . . . . . . . 23
Figure12. Effectsofxylanoticenzymesonxylan . . . . . . . . . . . . . . . . . . . . . . 24
Figure13. Generationofinhibitorsduringhardwoodhydrolysis . . . . . . . . . . . . 25
Figure14. Summary of the inhibition mechanisms of the toxic compounds in hemi-
cellulosichydrolysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Figure15. Inflowofweakacidinthecytosol . . . . . . . . . . . . . . . . . . . . . . . . 29
Figure16. Schematicrepresentationoftheuncouplingtheory . . . . . . . . . . . . . . 29
Figure17. Schematicrepresentationofsyntheticuncouplersaction . . . . . . . . . . . 29
Figure18. ModesoftoxicityoffurfuralinE.coli . . . . . . . . . . . . . . . . . . . . . . 32
Figure19. Overcomingtheinhibitionproblems . . . . . . . . . . . . . . . . . . . . . . 36
Figure20. Methodsforthedevelopmentofinhibitor-resistantmicroorganism . . . . . 41
Figure21. MetabolicpathwaysinE.coliBSS133 . . . . . . . . . . . . . . . . . . . . . . 43
Figure22. ExperimentalmatrixconstructedwithModde9.0 . . . . . . . . . . . . . . . 46
Figure23. Afermentationrun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Figure24. Evolution of the cell density for sodium acetate, furfural and POH for
differentinhibitorsconcentrations . . . . . . . . . . . . . . . . . . . . . . . . 51
Figure25. Evolutionofthecellviabilityforsodiumacetateandfurfuralfordifferent
inhibitorsconcentrations: . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Figure26. Atypicalfermentationprofile,initialconcentrations: AA:5g/L;Fur:1g/L;
POH:1g/L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Figure27. Celldensityduringthegrowthandproductionphases . . . . . . . . . . . . 57
Figure28. Evolutionofaceticacidthroughoutthefermentations . . . . . . . . . . . . 58
7
8 LISTOFFIGURES
Figure29. Plotofnormalizedresidualsforthethreeresponses . . . . . . . . . . . . . . 60
Figure30. Plotsofobservedagainstpredictedforthethreeresponses . . . . . . . . . 60
Figure31. Plotsofdistancetomodelforalltheresponses. . . . . . . . . . . . . . . . . 61
Figure32. Effectsplot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Figure33. Interactionplotsforfurfuralandaceticacid . . . . . . . . . . . . . . . . . . 62
Figure34. InteractionplotsforaceticacidandPOH . . . . . . . . . . . . . . . . . . . . 62
Figure35. InteractionplotsforfurfuralandPOH . . . . . . . . . . . . . . . . . . . . . 63
Figure36. Variableimportanceplot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Figure37. Response4Dcontourplot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Figure38. Response4Dcontourplot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Figure39. Response4Dcontourplot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Figure40. Effectplot(productionphase) . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Figure41. InteractionplotsforaceticacidandPOH(productionphase) . . . . . . . . 65
Figure42. VIPplot(productionphase) . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
List of Tables
Table1. Comparison of the fermentation performances of different succinic acid
producers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Table2. Comparisonofdifferenthydrolysismethods . . . . . . . . . . . . . . . . . . 24
Table3. Phenolsinhardwood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Table4. Compositionofdifferenthardwoodhemicellulosehydrolysates . . . . . . . 27
Table5. InhibitoreffectonE.coliLY01 . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Table6. Effects of acetic acid, furfural, POH and the combination acetic acid and
furfural on different parameters during ethanol fermentation by bakers’
yeast(asreportedin[74]). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Table7. CSLbasedmediumrecipe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Table8. PBSrecipe(pH=7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Table9. Comparison of inhibition (using OD ) on glucose and on xylose with
550
differentinhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Table10. Full factorial design: factors (acetic acid, furfural, and p-hydroxybenzoic
acid) and responses (specific growth rate, lag phase, biomass yield, volu-
metricproductivity,productionyieldandfinalsuccinicacidtitre) . . . . . . 56
Table11. Differencesbetweenthetworunsoftheexperimentnumber6(10g/Lacetic
acid,2g/LPOH). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Table12. PLStotalsummaryafterexclusion . . . . . . . . . . . . . . . . . . . . . . . . 59
Table13. Summaryoffitforindividualresponse . . . . . . . . . . . . . . . . . . . . . . 59
Table14. Sugarandinhibitorsconcentrationsduringthehydrolysatesfermentation . 66
Table15. InhibitionofE.coligrowthbyaceticacid,furfuralandPOH. . . . . . . . . . 69
9