Sustainable fungal biorefineries : optimizing production of valuable metabolites in oleaginous Mucoromycota

Abstract

Fungal biorefineries are important players in the emerging global bioeconomy and contribute to the transition from the traditional fossil-based production to a renewable, sustainable and environment-friendly bio-production. In such biorefineries, fermentation utilizing fungi as cell factories is a central process. Development of sustainable fungal biorefineries involves optimization of fungal fermentation for efficient feedstock utilization and high product yields. Since lignocellulosic biomass is sustainable and of high abundance, lignocellulose hydrolysates are considered as key carbon sources for large scale fungal fermentation. Mucoromycota filamentous fungi are powerful cell factories able to valorize hydrolyzed lignocellulose materials into a range of marketable products, such as lipids, biopolymers, pigments, proteins, enzymes and organic acids. Currently, the use of Mucoromycota for industrial production of fungal lipids for food, feed and biofuels applications is not economically viable. Therefore, a co-production concept has been proposed where several valuable bio-products can be produced in a single fungal fermentation process. This PhD work has focused on optimizing co-production of several metabolites in oleaginous Mucoromycota fungi by manipulation of growth media composition, with the ultimate goal of improving the economic sustainability of fungal biorefineries. The following media parameters were manipulated-type of nitrogen source, concentration of phosphorus substrate, and concentration of different metal ions. The optimization was performed using a high-throughput micro-cultivation system (Duetz- microtiter plate system) combined with different analytical techniques including vibrational spectroscopy. Total fungal lipid content was estimated either by gravimetry or gas chromatography (GC), while lipid profile was characterized by GC and nuclear magnetic resonance spectroscopy (NMR). NMR was also used for the characterization of phosphates in Mucoromycota biomass, in addition to estimation of total phosphorus by assay-based UV-visible (UV-VIS) spectroscopy. Fourier transform infrared spectroscopy (FTIR) and Fourier transform Raman spectroscopy (FT-Raman) were utilized for monitoring fungal fermentation in Duetz-MTPS and for biochemical fingerprinting of fungal biomass in order to measure the co-production of intracellular metabolites. The influence of two nitrogen sources (yeast extract and ammonium sulphate) and different amounts of phosphate substrate on the co-production of lipids, chitin/chitosan and polyphosphate, and on the lipid accumulation, in nine oleaginous Mucoromycota fungi was studied in Paper I and Paper II, respectively. To verify co-production, high-throughput FTIR spectroscopy was used as a main analytical method in Paper I. In Paper II, gas chromatography was used for analyzing the fatty acid profile and total lipid content was estimated gravimetrically. Strains with co-production potential and media components affecting the co-production and lipid accumulation were identified. In Paper III, the role of the metal ions calcium, copper, cobalt, iron, magnesium, manganese and zinc for growth of Mucor circinelloides was assessed. This strain was used since it is one of the most promising strains for the co-production of lipids, chitin/chitosan and polyphosphate. It was observed that calcium ions have a significant effect on the lipid accumulation in Mucor circinelloides. In order to investigate whether the effect of calcium ions is generally valid for other oleaginous Mucoromycota fungi, a study where six Mucoromycota fungi were grown in the presence and absence of calcium ions was performed in Paper V. Calcium availability was shown to affect lipid and polyphosphate accumulation under nonacidic conditions, while increased lipid accumulation was recorded mainly in acidic conditions lacking calcium ions. Analysis of fungal lipids was based on the Lewis method that utilizes simultaneous extraction and transesterification of lipids from the fungal biomass. However, since some Mucoromycota strains showed extraordinarily high lipid content while having strong cell wall structures hindering effective extraction, a modification of the Lewis method was developed in Paper IV. FTIR and FT-Raman spectroscopy were utilized for biochemical profiling of Mucoromycota biomass for revealing co-production of the targeted valuable metabolites, for monitoring fungal fermentation in Duetz-MTPS, and understanding the effect of the selected media components on Mucoromycota metabolism. In Paper VI, a comparison of the monitoring and biochemical profiling capacity of these two spectroscopies was investigated. Overall, this PhD work has provided knowledge on how manipulation of nitrogen source, phosphorus concentration and metal ions availability allow optimizing co-production in oleaginous Mucoromycota fungi. It was shown that several oleaginous Mucoromycota fungi have a great ability to perform co-production of triglyceride lipids, chitin/chitosan and polyphosphate biopolymers, and carotenoid pigments, and therefore have the potential to be powerful microbial cell factories in sustainable fungal biorefineries. The PhD work has contributed to the development of a more efficient and reliable lipid extraction method. Moreover, it has demonstrated how utilizing of modern vibrational spectroscopy techniques allows rapid and reliable optimization of media components for production of different metabolites and monitoring of fungal fermentations.

Bioraffinerier er viktige aktører i den kommende globale bioøkonomien og bidrar til overgangen fra tradisjonell fossilbasert produksjon til fornybar, bærekraftig og miljøvennlig bioproduksjon. I slike bioraffinerier er gjæring ved bruk av sopp som cellefabrikker en viktig prosess. Utvikling av bærekraftige soppbioraffinerier innebærer optimalisering av soppgjæring for effektiv råstoffutnyttelse og høye produktutbytter. Siden lignocellulose er et bærekraftig råstoff det finnes mye av, er lignocellulosehydrolysater en viktig potensiell karbonkilde for soppfermentering i storskala. Mucoromycota filamentøse sopp er effektive cellefabrikker som kan foredle hydrolyserte lignocellulosematerialer til en rekke salgbare produkter, som lipider, biopolymerer, pigmenter, proteiner, enzymer og organiske syrer. I dag er ikke bruken av Mucoromycota for industriell produksjon av sopplipider for mat-, fôr- og biodrivstoffanvendelser økonomisk. Derfor er det foreslått et samproduksjonskonsept hvor flere verdifulle bioprodukter kan produseres i en enkelt soppgjæringsprosess. Dette Doktorgradsarbeidet har fokuset ligget på å optimalisere produksjonen av flere metabolitter i oljerike Mucoromycota-sopp ved å manipulere sammensetningen av vekstmediene, med målet om å forbedre økonomien i soppbioraffinerier. Følgende parametere ble manipulert for å optimalisere vekstmediene: type nitrogenkilde, konsentrasjon av fosforsubstrat og konsentrasjon av forskjellige metallioner. Optimaliseringen ble utført ved hjelp av et mikrodyrkningssystem med stor kapasitet (Duetz-mikrotiterplatesystem) kombinert med forskjellige analytiske teknikker, inkludert vibrasjonsspektroskopi. Det totale innholdet av lipider i soppen ble estimert enten ved bruk av gravimetri eller gasskromatografi (GC), mens lipidprofilen ble bestemt ved bruk av GC og kjernemagnetisk resonansspektroskopi (NMR). NMR ble også brukt til analyse av polyfosfatinnhold i Mucoromycotabiomassen. Fourier transform infrarød spektroskopi (FTIR) og Fourier transform Raman spektroskopi (FT-Raman) ble brukt til å overvåke soppfermentering i Duetz-MTPS og for å få et biokjemisk fingeravtrykk av soppbiomasse for å måle samproduksjon av intracellulære metabolitter.