Products Components: Alcohols.

Alcohols (CnHn+2OH) are classified into primary, secondary, and tertiary alcohols, which can be branched or unbranched. They can also feature more than one OH-group (two OH-groups = diol; three OH-groups = triol). Presently, except for ethanol and sugar alcohols, they are mainly produced from fossil-based resources, such as petroleum, gas, and coal. Methanol and ethanol have the highest annual production volume accounting for 53 and 91 million tons/year, respectively. Most alcohols are used as fuels (e.g., ethanol), solvents (e.g., butanol), and chemical intermediates.This chapter gives an overview of recent research on the production of short-chain unbranched alcohols (C1-C5), focusing in particular on propanediols (1,2- and 1,3-propanediol), butanols, and butanediols (1,4- and 2,3-butanediol). It also provides a short summary on biobased higher alcohols (>C5) including branched alcohols.

Influence of the pH on the itaconic acid production with Aspergillus terreus.

Itaconic acid is mainly produced with the filamentous fungi Aspergillus terreus. An increase in the pH during the production phase of the cultivation resulted in an increase in the itaconic acid concentration. The pH was raised by a single pH shift ranging from pH 4 to 6 or by a pH control to pH 3. Different lyes can be used for the pH shift, but ammonia solution has proven to be the best, because here the productivity does not drop after the pH shift. The highest itaconic acid concentration of 146 g/L was reached when a pH control to pH 3 was started after 2.1 days of cultivation. This is an increase of 68 % to the cultivation without pH control. When this technique was combined with previously found optimizations, a final itaconic acid concentration of 129 g/L was reached after 4.7 days of cultivation, resulting in a productivity of 1.15 g/L/h.

Filamentous fungi in microtiter plates-an easy way to optimize itaconic acid production with Aspergillus terreus.

Itaconic acid is an important industrial building block and is produced by the filamentous fungi Aspergillus terreus. To make the optimization process more efficient, a scale-down from shake flasks to microtiter plates was performed. This resulted in comparable product formations, and 87.7 g/L itaconic acid was formed after 10 days of cultivation in the microtiter plate. The components of the minimal medium were varied independently for a media optimization. This resulted in an increase of the itaconic acid concentration by a variation of the KH2PO4 and CuSO4 concentrations. The cultivation with a higher KH2PO4 concentration in a 400-mL bioreactor showed an increase in the maximum productivity of 1.88 g/L/h, which was an increase of 74 % in comparison to the reference. Neither the phosphate concentration nor the nitrogen sources were limited at the start of the product formation. This showed that a limitation of these substances is not necessary for the itaconic acid formation.

Microbial production of itaconic acid: developing a stable platform for high product concentrations.

Biotechnologically produced itaconic acid (IA) is a promising organic acid with a wide range of applications and the potential to open up new application fields in the area of polymer chemistry, pharmacy, and agriculture. In this study, a systematic process optimization was performed with an own isolated strain of Aspergillus terreus and transferred from a 250-mL to a 15-L scale. An IA concentration of 86.2 g/L was achieved within 7 days with an overall productivity of 0.51 g/(L h), a maximum productivity of 1.2 g/(L h), and a yield of 86 mol%. A cultivation of other well-known A. terreus strains with the developed process showed no significant differences. Based on this, a process is developed providing a high final IA concentration independent of the used strain combined with high reproducibility.

High-level production of 1,3-propanediol from crude glycerol by Clostridium butyricum AKR102a.

The aim of this study was to optimize a biotechnological process for the production of 1,3-propanediol (1,3-PD) based on low-quality crude glycerol derived from biodiesel production. Clostridium butyricum AKR102a was used in fed-batch fermentations in 1-L and 200-L scale. The newly discovered strain is characterized by rapid growth, high product tolerance, and the ability to use crude glycerol at the lowest purity directly gained from a biodiesel plant side stream. Using pure glycerol, the strain AKR102 reached 93.7 g/L 1,3-PD with an overall productivity of 3.3 g/(L*h). With crude glycerol under the same conditions, 76.2 g/L 1,3-PD was produced with a productivity of 2.3 g/(L*h). These are among the best results published so far for natural producers. The scale up to 200 L was possible. Due to the simpler process design, only 61.5 g/L 1,3-PD could be reached with a productivity of 2.1 g/(L*h).

Production of high amounts of 3-hydroxypropionaldehyde from glycerol by Lactobacillus reuteri with strongly increased biocatalyst lifetime and productivity.

3-hydroxypropionaldehyde (3HPA) is a promising versatile substance derived from the renewable feedstock glycerol. It is a product of glycerol metabolism in Lactobacillus reuteri. Because of toxic effects, the biotechnological production is poor. In this work the biocatalyst lifetime and product formation could be drastically increased. In the established two-step process already applied, cells are grown in the first step under anaerobic conditions, and in the second step the immobilised or suspended biocatalyst is used for 3HPA-production under strict anaerobic conditions. In the first step it was possible to reach a biomass concentration of 5.5g CDW/L (OD(600)≈23.4). In the second step, normally, 3HPA accumulates to a toxic concentration and the reaction stops in less than 60min because of the interaction of 3HPA with cell components. To prevent this, the toxic product is bound to the newly found scavenger carbohydrazide to form the hydrazone. For the first time it was possible to recycle the immobilised biocatalyst for at least ten cycles (overall life time>33hours) in a repeated batch biotransformation with an overall production of 67g 3HPA. The optimal pH-value was between 6.8 and 7.2 at an optimal temperature of 40-45°C. In a single batch biotransformation with suspended resting cells it was possible to produce 150g/L 3HPA as carbohydrazone at an overall productivity of 10.7gL(-1)hours(-1). In a single fed-batch biotransformation at 45°C 138g/L glycerol was converted into 108g/L 3HPA with an overall productivity of 21.6gL(-1)hours(-1). This is the highest 3HPA concentration and productivities reported so far for the microbial production of 3HPA from glycerol.

An improved screening method for microorganisms able to convert crude glycerol to 1,3-propanediol and to tolerate high product concentrations.

A new screening method was developed and established to find high-performance bacteria for the conversion of crude glycerol to 1,3-propanediol. Three soil samples from palm oil-rich habitats were investigated using crude glycerol of a German biodiesel plant. Nine promising 1,3-propanediol producers could be found. Because of a special pH buffer system, a fast evaluation on microscale and high 1,3-propanediol concentrations up to 40 g L⁻¹ could be achieved. Three strains demonstrated very high product tolerance and were identified as Clostridium butyricum. Two strains, AKR91b and AKR102a, grew and produced 1,3-propanediol in the presence of 60 g L⁻¹ initial 1,3-propanediol, the strain AKR92a even in the presence of 77 g L⁻¹ 1,3-propanediol. The strains AKR91b and AKR102a tolerated up to 150 g L⁻¹ crude glycerol and produced 80% of the 1,3-propanediol attained from pure glycerol of the same concentration. Further criteria for the choice of a production strain were the pathogenicity (risk class), ability to grow on low-cost media, e.g., with less yeast extract, and robustness, e.g., process stability after several bioconversions. Overall, the strain C. butyricum AKR102a was chosen for further process optimization and scale-up due to its high productivity and high final concentration in a pH-regulated bioreactor.

Fermentation and microencapsulation of the nematophagous fungus Hirsutella rhossiliensis in a novel type of hollow beads.

In this work, fermentation and formulation aspects of the nematophagous fungus Hirsutella rhossiliensis BBA were investigated. When incubated in 2% (w/w) glucose and 0.5% (w/w) yeast extract medium in a 1-L Erlenmeyer flask without baffles, heavy pellet formation was observed. Only 40% of the mycelium had a size less than 500 µm. When a flask with three baffles was used, the portion of mycelium <500 µm rose to 95%. In the next step, the influence of aeration rate and stirrer speed on production of finely dispersed mycelium in a stirred tank reactor was investigated. The best fermentation results were obtained at 0.4 vvm and 400 rpm stirrer speed with 90% mycelium <500 µm and 5 g/L biomass. Then, mycelium was microencapsulated in hollow beads based on sulfoethylcellulose (SEC). Experiments on the capsule nutrient reservoir showed that 15% (w/w) corn gluten and 0.5% (w/w) yeast extract could be replaced with 3% (w/w) autoclaved baker's yeast which was never used as capsule additive before. Radial growth of mycelium out of dried hollow beads containing 1% (w/w) biomass and 3% (w/w) baker's yeast was faster than for alginate beads containing equivalent amounts of biomass and yeast indicating a higher bio-control potential.

Process for producing the potential food ingredient DFA III from inulin: screening, genetic engineering, fermentation and immobilisation of inulase II.

Difructose anhydride (DFA III) is a new potential sweet food additive. A screening was undertaken to isolate bacterial strains for conversion of inulin to DFA. Of special interest were thermotolerant enzymes. Some 400 strains were investigated, among four of them produce DFA and strain Buo141 expresses an extracellular enzyme which is stable at elevated temperatures. Based on metabolic data and 16S-rRNA-sequencing, the strain was identified as a new Arthrobacter species. For increased enzyme production, the inulase gene was cloned into E. coli XL1-blue, inulase II was expressed and its activity detected. After identifying the cleavage site, the sequence coding for a signal-peptide was eliminated from the plasmid and a beneficial amino acid exchange introduced by error-prone PCR. The recombinant E. coli was fermented to 10.5 g/l and after disruption an activity of 1.76 MioU/l was observed. The enzyme was flocculated from supernatant and entrapped in calcium alginate hydrogels. To enable production of uniform and small beads JetCutter technology was used with a production rate of 5600 beads/(snozzle). The influence of bead diameter on activity was investigated. An activity of 196 U/g was measured for 600-microm beads.