Valorization of a Pyrolytic Aqueous Condensate and Its Main Components for L-Malic Acid Production with Aspergillus oryzae DSM 1863

Pyrolytic aqueous condensate (PAC) might serve as a cost-effective substrate for microbial malic acid production, as it is an unused side stream of the fast pyrolysis of lignocellulosic biomass that contains acetol and acetate as potential carbon sources. In the present study, shake flask cultures were performed to evaluate the suitability of acetol and its combination with acetate as substrates for growth and L-malate production with the filamentous fungus Aspergillus oryzae. Acetol concentrations of up to 40 g/L were shown to be utilized for fungal growth. In combination with acetate, co-metabolization of both substrates for biomass and malate formation was observed, although the maximum tolerated acetol concentration decreased to 20 g/L. Furthermore, malate production on PAC detoxified by a combination of rotary evaporation, overliming and activated carbon treatment was studied. In shake flasks, cultivation using 100% PAC resulted in the production of 3.37 ± 0.61 g/L malate, which was considerably improved by pH adjustment up to 9.77 ± 0.55 g/L. A successful scale-up to 0.5-L bioreactors was conducted, achieving comparable yields and productivities to the shake flask cultures. Accordingly, fungal malate production using PAC was successfully demonstrated, paving the way for a bio-based production of the acid

Detoxification of a pyrolytic aqueous condensate from wheat straw for utilization as substrate in Aspergillus oryzae DSM 1863 cultivations

Background: The pyrolytic aqueous condensate (PAC) formed during the fast pyrolysis of wheat straw contains a variety of organic carbons and might therefore potentially serve as an inexpensive substrate for microbial growth. One of its main components is acetic acid, which was recently shown to be a suitable carbon source for the filamentous fungus Aspergillus oryzae. However, the condensate also contains numerous toxic compounds that inhibit fungal growth and result in a tolerance of only about 1%. Therefore, to enable the use of the PAC as sole substrate for A. oryzae cultivations, a pretreatment seems to be necessary. Results Various conditions for treatments with activated carbon, overliming, rotary evaporation and laccase were evaluated regarding fungal growth and the content of inhibitory model substances. Whereas the first three methods considerably increased the fungal tolerance to up to 1.625%, 12.5% and 30%, respectively, the enzymatic treatment did not result in any improvement. The optimum carbon load for the treatment with activated carbon was identified to be 10% (w/v) and overliming should ideally be performed at 100 °C and an initial pH of 12. The best detoxification results were achieved with rotary evaporation at 200 mbar as a complete removal of guaiacol and a strong reduction in the concentration of acetol, furfural, 2-cyclopenten-1-one and phenol by 84.9%, 95.4%, 97.7% and 86.2%, respectively, were observed. Subsequently, all possible combinations of the effective single methods were performed and rotary evaporation followed by overliming and activated carbon treatment proved to be most efficient as it enabled growth in 100% PAC shake-flask cultures and resulted in a maximum cell dry weight of 5.21 ± 0.46 g/L. Conclusion This study provides a comprehensive insight into the detoxification efficiency of a variety of treatment methods at multiple conditions. It was revealed that with a suitable combination of these methods, PAC toxicity can be reduced to such an extent that growth on pure condensate is possible. This can be considered as a first important step towards a microbial valorization of the pyrolytic side-stream with A. oryzae

Acetate as substrate for l-malic acid production with Aspergillus oryzae DSM 1863

Background Microbial malic acid production is currently not able to compete economically with well-established chemical processes using fossil resources. The utilization of inexpensive biomass-based substrates containing acetate could decrease production costs and promote the development of microbial processes. Acetate is a by-product in lignocellulosic hydrolysates and fast pyrolysis products or can be synthesized by acetogens during syngas fermentation. For the fermentation of these substrates, a robust microorganism with a high tolerance for biomass-derived inhibitors is required. Aspergillus oryzae is a suitable candidate due to its high tolerance and broad substrate spectrum. To pave the path towards microbial malic acid production, the potential of acetate as a carbon source for A. oryzae is evaluated in this study. Results A broad acetate concentration range was tested both for growth and malic acid production with A. oryzae. Dry biomass concentration was highest for acetic acid concentrations of 40–55 g/L reaching values of about 1.1 g/L within 48 h. Morphological changes were observed depending on the acetate concentration, yielding a pellet-like morphology with low and a filamentous structure with high substrate concentrations. For malic acid production, 45 g/L acetic acid was ideal, resulting in a product concentration of 8.44 ± 0.42 g/L after 192 h. The addition of 5–15 g/L glucose to acetate medium proved beneficial by lowering the time point of maximum productivity and increasing malic acid yield. The side product spectrum of cultures with acetate, glucose, and cultures containing both substrates was compared, showing differences especially in the amount of oxalic, succinic, and citric acid produced. Furthermore, the presence of CaCO$_{3}$, a pH regulator used for malate production with glucose, was found to be crucial also for malic acid production with acetate. Conclusions This study evaluates relevant aspects of malic acid production with A. oryzae using acetate as carbon source and demonstrates that it is a suitable substrate for biomass formation and acid synthesis. The insights provided here will be useful to further microbial malic acid production using renewable substrates

Optimization of carbon source efficiency for lipid production with the oleaginous yeast Saitozyma podzolica DSM 27192 applying automated continuous feeding

Background: Biotechnologically produced microbial lipids are of interest as potential alternatives for crude and plant oils. Their lipid profile is similar to plant oils and can therefore be a substitute for the production of biofuels, additives for food and cosmetics industry as well as building blocks for oleochemicals. Commercial microbial lipids production, however, is still not profitable and research on process optimization and cost reduction is required. This study reports on the process optimization using glucose or xylose with the unconventional oleaginous yeast Saitozyma podzolica DSM 27192 aiming to reduce the applied carbon source amount without sacrificing lipid productivity. Results: By optimizing the process parameters temperature and pH, lipid productivity was enhanced by 40%. Thereupon, by establishing a two-phase strategy with an initial batch phase and a subsequent fed-batch phase for lipid production in which a constant sugar concentration of about 10 g/L was maintained, resulted in saving of ~ 41% of total glucose and ~ 26% of total xylose. By performing the automated continuous sugar feed the total sugar uptake was improved to ~ 91% for glucose and ~ 92% for xylose and thus, prevented waste of unused carbon source in the cultivation medium. In addition, reduced glucose cultivation resulted in to 28% higher cell growth and 19% increase of lipid titer. By using xylose, the by-product xylonic acid was identified for the first time as by-product of S. podzolica. Conclusions: These findings provide a broad view of different cultivation process strategies with subsequent comparison and evaluation for lipid production with S. podzolica. Additionally, new biotechnological characteristics of this yeast were highlighted regarding the ability to produce valuable organic acids from sustainable and renewable sugars

Optimization of l-malic acid production from acetate with Aspergillus oryzae DSM 1863 using a pH-coupled feeding strategy

Background Malic acid, a dicarboxylic acid mainly used in the food industry, is currently produced from fossil resources. The utilization of low-cost substrates derived from biomass could render microbial processes economic. Such feedstocks, like lignocellulosic hydrolysates or condensates of fast pyrolysis, can contain high concentrations of acetic acid. Acetate is a suitable substrate for L-malic acid production with the filamentous fungus Aspergillus oryzae DSM 1863, but concentrations obtained so far are low. An advantage of this carbon source is that it can be used for pH control and simultaneous substrate supply in the form of acetic acid. In this study, we therefore aimed to enhance L-malate production from acetate with A. oryzae by applying a pH-coupled feeding strategy. Results In 2.5-L bioreactor fermentations, several feeding strategies were evaluated. Using a pH-coupled feed consisting of 10 M acetic acid, the malic acid concentration was increased about 5.3-fold compared to the batch process without pH control, resulting in a maximum titer of 29.53 ± 1.82 g/L after 264 h. However, it was not possible to keep both the pH and the substrate concentration constant during this fermentation. By using 10 M acetic acid set to a pH of 4.5, or with the repeated addition of NaOH, the substrate concentration could be maintained within a constant range, but these strategies did not prove beneficial as lower maximum titers and yields were obtained. Since cessation of malic acid production was observed in later fermentation stages despite carbon availability, a possible product inhibition was evaluated in shake flask cultivations. In these experiments, malate and succinate, which is a major by-product during malic acid production, were added at concentrations of up to 50 g/L, and it was found that A. oryzae is capable of organic acid production even at high product concentrations. Conclusions This study demonstrates that a suitable feeding strategy is necessary for efficient malic acid production from acetate. It illustrates the potential of acetate as carbon source for microbial production of the organic acid and provides useful insights which can serve as basis for further optimization

Enzymatic synthesis of glucose monodecanoate in a hydrophobic deep eutectic solvent

Environmentally friendly and biodegradable reaction media are an important part of a sustainable glycolipid production in the transition to green chemistry. Deep eutectic solvents (DESs) are an ecofriendly alternative to organic solvents. So far, only hydrophilic DESs were considered for enzymatic glycolipid synthesis. In this study, a hydrophobic DES consisting of (-)-menthol and decanoic acid is presented for the first time as an alternative to hydrophilic DES. The yields in the newly introduced hydrophobic DES are significantly higher than in hydrophilic DESs. Different reaction parameters were investigated to optimize the synthesis further. Twenty milligrams per milliliter iCalB and 0.5 M glucose resulted in the highest initial reaction velocity for the esterification reaction, while the highest initial reaction velocity was achieved with 1.5 M glucose in the transesterification reaction. The enzyme was proven to be reusable for at least five cycles without significant loss of activity

Enhanced l-Malic Acid Production by Aspergillus oryzae DSM 1863 Using Repeated-Batch Cultivation

l-Malic acid is a C4-dicarboxylic acid and a potential key building block for a bio-based economy. At present, malic acid is synthesized petrochemically and its major market is the food and beverages industry. In future, malic acid might also serve as a building block for biopolymers or even replace the commodity chemical maleic anhydride. For a sustainable production of l-malic acid from renewable resources, the microbial synthesis by the mold Aspergillus oryzae is one possible route. As CO2 fixation is involved in the biosynthesis, high yields are possible, and at the same time greenhouse gases can be reduced. In order to enhance the production potential of the wild-type strain Aspergillus oryzae DSM 1863, process characteristics were studied in shake flasks, comparing batch, fed-batch, and repeated-batch cultivations. In the batch process, a prolonged cultivation time led to malic acid consumption. Keeping carbon source concentration on a high level by pulsed feeding could prolong cell viability and cultivation time, however, did not result in significant higher product levels. In contrast, continuous malic acid production could be achieved over six exchange cycles and a total fermentation time of 19 days in repeated-batch cultivations. Up to 178 g/L l-malic acid was produced. The maximum productivity (0.90 ± 0.05 g/L/h) achieved in the repeated-batch cultivation had more than doubled than that achieved in the batch process and also the average productivity (0.42 ± 0.03 g/L/h for five exchange cycles and 16 days) was increased considerably. Further repeated-batch experiments confirmed a positive effect of regular calcium carbonate additions on pH stability and malic acid synthesis. Besides calcium carbonate, nitrogen supplementation proved to be essential for the prolonged malic acid production in repeated-batch. As prolonged malic acid production was only observed in cultivations with product removal, product inhibition seems to be the major limiting factor for malic acid production by the wild-type strain. This study provides a systematic comparison of different process strategies under consideration of major influencing factors and thereby delivers important insights into natural l-malic acid production

Caproate production from Enset fiber in one-pot two-step fermentation using anaerobic fungi (Neocallimastix cameroonii strain G341) and Clostridium kluyveri DSM 555

Lignocellulosic biomass plays a crucial role in creating a circular bioeconomy and minimizing environmental impact. Enset biomass is a byproduct of traditional Ethiopian Enset food processing that is thrown away in huge quantities. This study aimed to produce caproate from Enset fiber using Neocallimastix cameroonii strain G$_{341}$ and Clostridium kluyveri DSM $_{555}$ in one-pot two-step fermentation

Draft Genome Sequence of the Oleaginous Yeast Apiotrichum porosum (syn. Trichosporon porosum) DSM 27194

Here, we present the draft genome sequence of Apiotrichum porosum DSM 27194 generated on PacBio platform. Characterization of this oleaginous yeast originally collected from the grassland in Karlsruhe Germany, revealed potential for its utilization as a source of single cell oil (SCO) and gluconic acid (GA). The availability of the genome sequence provides a valuable resource for the elucidation of the genetic processes determining SCO and GA biosynthesis