Producing organic acids from pomace wastes – a biorefinery concept

Thesis (PhD)--Stellenbosch University, 2022.ENGLISH SUMMARY: The production of energy and valuable products via petroleum refineries could be partially replaced by biorefineries that are utilising renewable biomass as substrate. For example, an important metabolite such as malic acid in fruit wastes, like apple and grape pomace, could serve as a substrate for microbial conversion to organic acids and other green chemicals. Specific waste sources were investigated to isolate native yeast strains that may have acquired improved malic acid degradation abilities. The 98 new isolates from grape, apple and plum waste were screened for their ability to degrade extracellular malic acid relative to 50 known strains. Most (94%) of the new isolates degraded more than 50% of the malic acid in both the presence and absence of glucose, whereas only 14% of the known strains could do so, thus confirming the value of exploring and exploiting natural biodiversity for new candidates. The eight best isolates were evaluated in synthetic media, with two strains showing potential for the production of ethanol and acetic acid during aerobic and oxygen-limited growth on apple and grape pomace. The screening of yeasts led to the identification of Saccharomyces cerevisiae strain 61 that showed stronger malic acid-degrading capabilities than expected for this species. Preliminary characterisation revealed that strain 61 degraded malic acid in synthetic media more efficiently (potential for application in the wine industry), produced significantly higher biomass (potential for recombinant protein expression in S. cerevisiae) and is more heat-resistant (potential for consolidated bioprocessing) than commercial wine yeasts. Recombinant S. cerevisiae strains were constructed to express either the Candida krusei (Ckr_fum) or the codon-optimised Escherichia coli (Eco_fum) fumarase gene with or without the XYNSEC secretion signal of Trichoderma reesei xylanase 2. These strains were further engineered to co-express the Schizosaccharomyces pombe transporter (mae1) gene for the active uptake of malic acid. Both strains 5A and 31B(p1), expressing the Ckr_fum and mae1 genes [31B(p1) also contains the XYNSEC secretion signal], produced fumaric acid from extracellular malic acid. Our findings illustrate that disruption of the natural FUM1 gene in S. cerevisiae strains is beneficial when using malic acid as a substrate for fumaric acid production. The inclusion of a transporter allowed for better malic acid degradation and quicker fumaric acid production, and the yeast fumarase was more effective than the bacterial fumarase for fumaric acid production. Strain 5A was able to produce 0.065 g/L fumaric and 2.55 g/L ethanol after 72 h on grape pomace, suggesting that this strain may have potential application for a fruit waste biorefinery following optimisations. This study resulted in the isolation of a number of yeast strains with the ability to utilise malic acid, including S. cerevisiae strain 61. After unravelling the reason(s) for its enhanced malic acid utilisation, this strain could be a promising candidate for the development of a fumaric acid-producing biorefinery host strain. However, this would require the construction of a recombinant strain expressing an effective fumarase and dicarboxylic acid transporter, with the Ckr_fum and mae1 genes as potential candidates.AFRIKAANS OPSOMMING: Die produksie van energie en waardevolle produkte deur middel van petroleum raffinaderye kan gedeeltelik deur bioraffinaderye vervang word wat hernubare biomassa as substraat benut. Byvoorbeeld, ‘n belangrike metaboliet soos appelsuur wat in vrugte-afval soos appel- en druiwereste voorkom, kan as substraat gebruik word vir mikrobiese omskakeling na organiese sure en ander groen chemikalieë. Spesifieke afvalbronne is ondersoek om inheemse gisrasse met beter appelsuur-afbraakvermoë te isoleer. Die 98 nuwe isolate vanuit druiwe, appel en pruimreste se vermoë om ekstrasellulêre appelsuur af te breek is met 50 bekende gisstamme vergelyk. Die meeste (94%) van die nuwe isolate kon meer as 50% appelsuur in beide die teenwoordigheid en afwesigheid van glukose afbreek, terwyl slegs 14% van die bekende stamme dieselfde kon doen. Dit bevestig dat die ondersoek en ontginning van natuurlike biodiversiteit waardevol kan wees vir die isolasie van nuwe kandidate. Die agt beste isolate is in sintetiese media geëvalueer, met twee stamme wat potensiaal vir die produksie van etanol en asynsuur tydens aërobiese en suurstofbeperkte groei op appel- en druiwereste getoon het. Die sifting van giste het gelei tot die identifisering van Saccharomyces cerevisiae stam 61 met 'n sterker vermoë vir die afbraak van appelsuur as wat vir hierdie spesie verwag word. Voorlopige karakterisering het aangedui dat stam 61 appelsuur in sintetiese media meer doeltreffend kon afbreek (potensiaal vir toepassing in die wynbedryf), aansienlik hoër biomassa produseer (potensiaal vir rekombinante proteïenuitdrukking toepassings in S. cerevisiae) en meer hittebestand (potensiaal vir gekonsolideerde bioprosessering) as kommersiële wyngiste was. Rekombinante S. cerevisiae-stamme is gekonstrueer om die Candida krusei (Ckr_fum) of die kodon-geoptimaliseerde Escherichia coli (Eco_fum) fumarasegeen met of sonder die XYNSEC sekresiesein van Trichoderma reesei xylanase 2 uit te druk. Hierdie rasse is verder aangepas vir mede-uitdrukking van die Schizosaccharomyces pombe (mae1) malaattransportergeen vir aktiewe opname van appelsuur. Die 5A en 31B(p1) stamme wat die Ckr_fum en mae1 gene uitdruk [31B(p1) bevat ook die XYNSEC sekresiesein], kon fumaarsuur uit ekstrasellulêre appelsuur produseer. Ons bevindinge illustreer dat ontwrigting van die natuurlike FUM1-geen in S. cerevisiae-stamme voordelig is wanneer appelsuur as 'n substraat vir fumaarsuurproduksie gebruik word. Die insluiting van 'n transportergeen het tot beter appelsuurafbraak en vinniger fumaarsuurproduksie gelei, en die gis-fumarase was meer effektief vir fumaraatproduksie as die bakteriële fumarase. Stam 5A het 0,065 g/L fumaarsuur en 2,55 g/L etanol na 72 uur se groei op druiweafval geproduseer, wat aandui dat hierdie gisras moontlike toepassings in 'n vrugteafval bioraffinadery kan vind na die afloop van verdere verbeteringe. Hierdie studie het gelei tot die isolasie van ʼn aantal gisstamme met die vermoë om malaatsuur te benut, insluitende S. cerevisiae stam 61. Na ontrafeling van die meganismes(s) vir sy verbeterde appelsuurbenutting, kan hierdie stam 'n belowende kandidaat vir die ontwikkeling van 'n fumaarsuur-produserende bioraffinaderygasheer wees. Dit sal egter die konstruksie van ʼn rekombinante stam benodig wat ʼn effektiewe fumarase en dikarboksielsuur- transporter uitdruk, met die Ckr_fum en mae1 gene as potensiële kandidate.Doctora