Improving protein stability is an important goal for basic research as well as for clinical and industrial applications but no commonly accepted and widely used strategy for efficient engineering is known. Beside random approaches like error prone PCR or physical techniques to stabilize proteins, e.g. by immobilization, in silico approaches are gaining more attention to apply target-oriented mutagenesis. In this review different algorithms for the prediction of beneficial mutation sites to enhance protein stability are summarized and the advantages and disadvantages of FoldX are highlighted. The question whether the prediction of mutation sites by the algorithm FoldX is more accurate than random based approaches is addressed.

Polyunsaturated fatty acids (PUFAs) of the ω-3 and ω-6 class (e.g., α-linolenic acid, linoleic acid) are essential for maintaining biofunctions in mammalians like humans. Due to the fact that humans cannot synthesize these essential fatty acids, they must be taken up from different food sources. Classical sources for these fatty acids are porcine liver and fish oil. However, microbial lipids or single cell oils, produced by oleaginous microorganisms such as algae, fungi and bacteria, are a promising source as well. These single cell oils can be used for many valuable chemicals with applications not only for nutrition but also for fuels and are therefore an ideal basis for a bio-based economy. A crucial point for the establishment of microbial lipids utilization is the cost-effective production and purification of fuels or products of higher value. The fermentative production can be realized by submerged (SmF) or solid state fermentation (SSF). The yield and the composition of the obtained microbial lipids depend on the type of fermentation and the particular conditions (e.g., medium, pH-value, temperature, aeration, nitrogen source). From an economical point of view, waste or by-product streams can be used as cheap and renewable carbon and nitrogen sources. In general, downstream processing costs are one of the major obstacles to be solved for full economic efficiency of microbial lipids. For the extraction of lipids from microbial biomass cell disruption is most important, because efficiency of cell disruption directly influences subsequent downstream operations and overall extraction efficiencies. A multitude of cell disruption and lipid extraction methods are available, conventional as well as newly emerging methods, which will be described and discussed in terms of large scale applicability, their potential in a modern biorefinery and their influence on product quality. Furthermore, an overview is given about applications of microbial lipids or derived fatty acids with emphasis on food applications.

Malic acid derived from fossil resources is currently applied in the food and beverage industries with a medium global production capacity. However, in the transition from a fossil-based to a bio-based economy, biotechnologically produced L-malic acid may become an important platform chemical with many new applications, especially in the field of biopolymers. In this review, currently used petrochemical production routes to DL-malic acid are outlined and insights into possible bio-based alternatives for microbial L-malic acid production are provided. Besides ecological reasons, the possibility to produce enantiopure L-malic acid by microbial fermentation is the biggest advantage over chemical synthesis. State-of-the-art and open challenges concerning production host engineering, substrate choice and downstream processing are addressed. With regard to production hosts, a literature overview is given covering the leading natural production strains of Aspergillus, Ustilago and Aureobasidium, as well as Escherichia coli as the most important engineered recombinant host. The utilization of renewable substrates as an alternative to glucose is emphasized in particular as a key aspect for a competitive bio-based production. Out of the alternative substrates discussed in this review, the industrial side-streams crude glycerol and molasses seem to be most promising for large-scale L-malic acid production.

The yeast strains Cryptococcus podzolicus, Trichosporon porosum and Pichia segobiensis were isolated from soil samples and identified as oleaginous yeast strains beneficial for the establishment of microbial production processes for sustainable lipid production suitable for several industrial applications. When cultured in bioreactors with glucose as the sole carbon source C. podzolicus yielded 31.8% lipid per dry biomass at 20°C, while T. porosum yielded 34.1% at 25°C and P. segobiensis 24.6% at 25°C. These amounts correspond to lipid concentrations of 17.97 g/L, 17.02 g/L and 12.7 g/L and volumetric productivities of 0.09 g/Lh, 0.1 g/Lh and 0.07 g/Lh, respectively. During the culture of C. podzolicus 30 g/l gluconic acid was detected as by-product in the culture broth and 12 g/L gluconic acid in T. porosum culture. The production of gluconic acid was eliminated for both strains when glucose was substituted by xylose as the carbon source. Using xylose lipid yields were 11.1 g/L and 13.9 g/L, corresponding to 26.8% and 33.4% lipid per dry biomass and a volumetric productivity of 0.07 g/Lh and 0.09 g/Lh, for C. podzolicus and T. porosum respectively. The fatty acid profile analysis showed that oleic acid was the main component (39.6 to 59.4%) in all three strains and could be applicable for biodiesel production. Palmitic acid (18.4 to 21.1%) and linolenic acid (7.5 to 18.7%) are valuable for cosmetic applications. P. segobiensis had a considerable amount of palmitoleic acid (16% content) and may be suitable for medical applications.

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Synthesis gas (syngas) fermentation using acetogenic bacteria is an approach for production of bulk chemicals like acetate, ethanol, butanol, or 2,3-butandiol avoiding the fuel vs. food debate by using carbon monoxide, carbon dioxide, and hydrogen from gasification of biomass or industrial waste gases. Suffering from energetic limitations, yields of C4-molecules produced by syngas fermentation are quite low compared with ABE fermentation using sugars as a substrate. On the other hand, fungal production of malic acid has high yields of product per gram metabolized substrate but is currently limited to sugar containing substrates. In this study, it was possible to show that Aspergilus oryzae is able to produce malic acid using acetate as sole carbon source which is a main product of acetogenic syngas fermentation. Bioreactor cultivations were conducted in 2.5 L stirred tank reactors. During the syngas fermentation part of the sequential mixed culture, Clostridium ljungdahlii was grown in modified Tanner medium and sparged with 20 mL/min of artificial syngas mimicking a composition of clean syngas from entrained bed gasification of straw (32.5 vol-% CO, 32.5 vol-% H2, 16 vol-% CO2, and 19 vol-% N2) using a microsparger. Syngas consumption was monitored via automated gas chromatographic measurement of the off-gas. For the fungal fermentation part gas sparging was switched to 0.6 L/min of air and a standard sparger. Ammonia content of medium for syngas fermentation was reduced to 0.33 g/L NH4Cl to meet the requirements for fungal production of dicarboxylic acids. Malic acid production performance of A. oryzae in organic acid production medium and syngas medium with acetate as sole carbon source was verified and gave YP∕S values of 0.28 g/g and 0.37 g/g respectively. Growth and acetate formation of C. ljungdahlii during syngas fermentation were not affected by the reduced ammonia content and 66 % of the consumed syngas was converted to acetate. The overall conversion of CO and H2 into malic acid was calculated to be 3.5 g malic acid per mol of consumed syngas or 0.22 g malic acid per gram of syngas.

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L-Malic acid and fumaric acid are C4 dicarboxylic organic acids and considered as promising chemical building blocks. They can be applied as food preservatives and acidulants in rust removal and as polymerization starter units. Molds of the genus Aspergillus are able to produce malic acid in large quantities from glucose and other carbon sources. In order to enhance the production potential of Aspergillus oryzae DSM 1863, production and consumption rates in an established bioreactor batch-process based on glucose were determined. At 35 °C, up to 42 g/L malic acid was produced in a 168-h batch process with fumaric acid as a by-product. In prolonged shaking flask experiments (353 h), the suitability of the alternative carbon sources xylose and glycerol at a carbon-to-nitrogen (C/N) ratio of 200:1 and the influence of different C/N ratios in glucose cultivations were tested. When using glucose, 58.2 g/L malic acid and 4.2 g/L fumaric acid were produced. When applying xylose or glycerol, both organic acids are produced but the formation of malic acid decreased to 45.4 and 39.4 g/L, respectively. Whereas the fumaric acid concentration was not significantly altered when cultivating with xylose (4.5 g/L), it is clearly enhanced by using glycerol (9.3 g/L). When using glucose as a carbon source, an increase or decrease of the C/N ratio did not influence malic acid production but had an enormous influence on fumaric acid production. The highest fumaric acid concentrations were determined at the highest C/N ratio (300:1, 8.44 g/L) and lowest at the lowest C/N ratio (100:1, 0.7 g/L).