Pilot-scale production of mcl-PHA by Pseudomonas citronellolis using acetic acid as the sole carbon source.

Medium-chain-length polyhydroxyalkanoates (mcl-PHA) are biobased materials with promising properties for environmentally friendly applications. Due to high production costs, which are related to the cost of the carbon sources combined with conversion insufficiencies, currently only small quantities are produced. This results in a lack of reliable data on properties and application potential for the variety of polymers from different types of production strains. This study investigated the potential for the production of mcl-PHA from volatile fatty acids (VFA) at a larger scale, given their potential as low-cost and sustainable raw material within a carboxylate-platform based biorefinery. Pseudomonas citronellolis (DSMZ 50332) was chosen as the production strain, and acetic acid was selected as the main carbon and energy source. Nitrogen was limited to trigger polymer production, and a fed-batch process using a pH-stat feeding regime with concentrated acid was established. We report successful production, extraction, and characterization of mcl PHA, obtaining a total of 1.76 kg from two 500-litre scale fermentations. The produced polymer was identified as a copolymer of 3-hydroxydecanoate (60.7%), 3-hydroxyoctanoate (37.3%), and 3-hydroxyhexanoate (2.0%) with a weight average molecular weight (Mw) of 536 kDa. NMR analysis indicates the presence of unsaturated side chains, which may offer additional possibilities for modification. The results confirm that there is a potential to produce significant amounts of mcl-PHA with interesting rubber-like properties from waste-derived VFA.

Comparative assessment of environmental impacts of 1st generation (corn feedstock) and 3rd generation (carbon dioxide feedstock) PHA production pathways using life cycle assessment.

Polyhydroxyalkanoates (PHA) are bio-based and biodegradable alternatives to conventional plastic types and have the potential to reduce the environmental impacts along the life cycle. In comparison to already established production routes for PHA (heterotrophic production) based on renewable feedstock like glucose (first generation feedstock), novel production routes, such as the photoautotrophic production of PHA based on CO2 as feedstock (third generation feedstock) could offer new perspectives with regard to the reduction in the environmental impacts. To quantify the environmental impacts of PHA produced via photoautotrophic and heterotrophic production pathways, life cycle assessment (LCA) methodology based on ISO 14040/44 was applied, thus conducting a first of its kind comparative study for PHA based on third generation feedstock. The results show that the photoautotrophic production of PHA has advantages in comparison to heterotrophic PHA based on glucose originating from corn as feedstock in all the assessed environmental impact categories, thus showing the environmental potential of novel production routes for bioplastics. Additionally, the results of the LCA show that the chloroform-based extraction method, commonly used in the downstream processes of both the technologies, has a significant contribution of environmental impacts in the production of PHA. Therefore, the reduction of chloroform loss during the extraction process can reduce its environmental impact. Our results indicate that PHA production from CO2 using the photoautotrophic production route is a promising technology with regard to the environmental impacts when compared to the heterotrophic production based on glucose feedstock.

Advances and trends in microbial production of polyhydroxyalkanoates and their building blocks.

With the rapid development of synthetic biology, a variety of biopolymers can be obtained by recombinant microorganisms. Polyhydroxyalkanoates (PHA) is one of the most popular one with promising material properties, such as biodegradability and biocompatibility against the petrol-based plastics. This study reviews the recent studies focusing on the microbial synthesis of PHA, including chassis engineering, pathways engineering for various substrates utilization and PHA monomer synthesis, and PHA synthase modification. In particular, advances in metabolic engineering of dominant workhorses, for example Halomonas, Ralstonia eutropha, Escherichia coli and Pseudomonas, with outstanding PHA accumulation capability, were summarized and discussed, providing a full landscape of diverse PHA biosynthesis. Meanwhile, we also introduced the recent efforts focusing on structural analysis and mutagenesis of PHA synthase, which significantly determines the polymerization activity of varied monomer structures and PHA molecular weight. Besides, perspectives and solutions were thus proposed for achieving scale-up PHA of low cost with customized material property in the coming future.

Cell Retention as a Viable Strategy for PHA Production from Diluted VFAs with Bacillus megaterium.

The production of biodegradable and biocompatible materials such as polyhydroxyalkanoates (PHAs) from waste-derived volatile fatty acids (VFAs) is a promising approach towards implementing a circular bioeconomy. However, VFA solutions obtained via acidification of organic wastes are usually too diluted for direct use in standard batch or fed-batch processes. To overcome these constraints, this study introduces a cell recycle fed-batch system using Bacillus megaterium uyuni S29 for poly(3-hydroxybutyrate) (P3HB) production from acetic acid. The concentrations of dry cell weight (DCW), P3HB, acetate, as well as nitrogen as the limiting substrate component, were monitored during the process. The produced polymer was characterized in terms of molecular weight and thermal properties after extraction with hypochlorite. The results show that an indirect pH-stat feeding regime successfully kept the strain fed without prompting inhibition, resulting in a dry cell weight concentration of up to 19.05 g/L containing 70.21% PHA. After appropriate adaptations the presented process could contribute to an efficient and sustainable production of biopolymers.

The influence of different carbon sources on growth and single cell oil production in oleaginous yeasts Apiotrichum brassicae and Pichia kudriavzevii.

Oleaginous yeasts offer an interesting possibility for renewable lipid production, since the single cell oil accumulated can be based on a wide range of cheap, waste-derived carbon sources. Here, several short chain carboxylic acids and sugars commonly found in these substrates were assessed as carbon sources for Apiotrichum brassicae and Pichia kudriavzevii. While both strains were able to utilize all carbon sources employed, high volumetric lipid productivities (0.4 g/Lh) and lipid contents (68%) could be reached particularly with acetic acid as carbon source. Odd-numbered volatile fatty acids led to lower productivities and lipid contents, but the lipids contained unusually high proportions of odd-numbered fatty acids (up to 80% of total fatty acids). These fatty acids are rather uncommon in nature and might offer the possibility for various high value applications. In conclusion both strains are able to utilize a wide range of substrates potentially present in waste-derived substrates. Lipid content and volumetric lipid productivity strongly depend on the carbon source, with even-numbered volatile fatty acids resulting in the highest values. For volatile fatty acids in particular, the carbon source also strongly influences the composition of the lipids produced by the yeast strains.

Waste-derived volatile fatty acids as carbon source for added-value fermentation approaches.

The establishment of a sustainable circular bioeconomy requires the effective material recycling from biomass and biowaste beyond composting/fertilizer or anaerobic digestion/bioenergy. Recently, volatile fatty acids attracted much attention due to their potential application as carbon source for the microbial production of high added-value products. Their low-cost production from different types of wastes through dark fermentation is a key aspect, which will potentially lead to the sustainable production of fuels, materials or chemicals, while diminishing the waste volume. This article reviews the utilization of a volatile fatty acid platform for the microbial production of polyhydroxyalkanoates, single cell oil and omega-3 fatty acids, giving emphasis on the fermentation challenges for the efficient implementation of the bioprocess and how they were addressed. These challenges were addressed through a research project funded by the European Commission under the Horizon 2020 programme entitled 'VOLATILE-Biowaste derived volatile fatty acid platform for biopolymers, bioactive compounds and chemical building blocks'.

Effects of nutrient and oxygen limitation, salinity and type of salt on the accumulation of poly(3-hydroxybutyrate) in Bacillus megaterium uyuni S29 with sucrose as a carbon source.

Due to high manufacturing costs, industrial production and application of bio-based polyhydroxyalkanoates (PHA) as bioplastics remain below the expected potential. Improving yields and productivities during biotechnological production will contribute to eliminating existing shortcomings and should therefore be a priority in process development with new strains and substrates. The present study investigates key parameters such as different nutrient and oxygen limitation strategies and the salinity and type of salt to determine their impact on growth and poly(3-hydroxybutyrate) (P(3HB)) formation behaviour of Bacillus megaterium. The oxygen-limiting conditions applied resulted in a longer process duration and were found to be least effective with regard to P(3HB) content in the biomass. A higher P(3HB) content of 0.42 g g-1 was achieved when nitrogen was limited compared to 0.34 g g-1 under phosphate-limiting conditions; however, sucrose utilization was better when phosphate was limited. Replacing NaCl by KCl and evaluating different concentrations ranging from 0.08 to 1.7 mol L-1 in the process medium showed that B. megaterium has a higher tolerance to KCl as the biomass and P(3HB) formation was increased to 0.48 g g-1 compared to 0.36 g g-1. The combination of applying KCl instead of NaCl together with phosphorous limitation significantly increased P(3HB) productivity to 0.25 g L-1 h-1 compared to 0.09 g L-1 h-1. It can be concluded that the effective utilization of sucrose as a carbon source requires a combination of high nitrogen and low phosphorous concentration and a salt content of 0.6 g L-1 KCl for P(3HB) production with B. megaterium uyuni S29.

High cell-density production of poly(3-hydroxybutyrate) in a membrane bioreactor.

Agro-industrial residues with a low carbon content, such as whey, stillage or wastewater from plant oil mills are abundant and cheap. However, they cannot be used directly in highly productive industrial poly(3-hydroxybutyrate) (P3HB) production, as the classical fed-batch fermentation strategy requires highly concentrated feed streams. This problem has been circumvented in this report by retaining the cells during the fermentation in the bioreactor using an external microfiltration module. Synthetic medium containing a glucose concentration of 50g/L was continuously fed to Cupriavidus necator, which converted the sugar to P3HB. With this setup we were able to achieve high productivities (3.10g P3HB/(Lh)) and reach high cell densities (148g/L) containing 76% P3HB, and obtained good yields (0.33g P3HB/g added glucose). The added sugar from the feed was instantly consumed by the bacteria, resulting in a negligible loss of sugar to the permeate. This approach creates the possibility of polyhydroxyalkanoate production from a range of cheap and easily available substrates, for which only waste water treatment or biogas production has been cost-competitive until now.

Direct fermentation of potato starch and potato residues to lactic acid by Geobacillus stearothermophilus under non-sterile conditions.

BACKGROUND: Lactic acid is an important biorefinery platform chemical. The use of thermophilic amylolytic microorganisms to produce lactic acid by fermentation constitutes an efficient strategy to reduce operating costs, including raw materials and sterilization costs. RESULTS: A process for the thermophilic production of lactic acid by Geobacillus stearothermophilus directly from potato starch was characterized and optimized. Geobacillus stearothermophilus DSM 494 was selected out of 12 strains screened for amylolytic activity and the ability to form lactic acid as the major product of the anaerobic metabolism. In total more than 30 batches at 3-l scale were run at 60 °C under non-sterile conditions. The process developed produced 37 g L-1 optically pure (98%) L-lactic acid in 20 h from 50 g L-1 raw potato starch. As co-metabolites smaller amounts (<7% w/v) of acetate, formate and ethanol were formed. Yields of lactic acid increased from 66% to 81% when potato residues from food processing were used as a starchy substrate in place of raw potato starch. CONCLUSIONS: Potato starch and residues were successfully converted to lactic acid by G. stearothermophilus. The process described in this study provides major benefits in industrial applications and for the valorization of starch-rich waste streams. © 2015 The Authors.Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Wheat bran biorefinery--an insight into the process chain for the production of lactic acid.

The present study investigates a wheat bran biorefinery including major processing steps pre-extraction, pre-treatment and lactic acid fermentation. Concerning the dry mass distribution, during the pre-extraction 32% of the feedstock was recovered, offering some perspectives for applications in food area. The pre-treatment (hydrothermal/enzymatic hydrolysis) of the remaining cake solubilized 34% of dry mass and led to a fermentable sugar concentration of 21g/L. The fermentation resulted in a lactic acid yield of 0.73g/g substrate. Concentrating the fermentation feed via nanofiltration did not improve the lactic acid productivity. Taking into account that Lactobacillus pentosus, a heterofermentative microorganism was used, the dry mass balance revealed a product yield of 47% (32% extract, 15% lactic acid). Based on a theoretical consideration involving a cellulolytic enzyme production (10% feedstock allocation) and lignin utilization, under optimized conditions a maximum product yield of around 80% (35% extract, 39% lactic acid, 6% lignin) could be expected.

Effect of wood ash treatment on improving the fermentability of wood hydrolysate.

Softwood hydrolysates were overlimed with wood ash to improve the fermentability of hydrolysates. It could be demonstrated in fermentation tests that wood ash treatment increases fermentability compared to the hydrolysates untreated and treated with alkaline compounds such as Ca(OH)(2), NaOH, and KOH, which are commonly used for overliming. The enhanced fermentability of the hydrolysate treated with wood ash is due to the reduction of the inhibitors of the fermentation such as furan and phenolic compounds and to nutrient effects of some inorganic components from the wood ash on the fermentation.

Dilute-acid hydrolysis of sugarcane bagasse at varying conditions.

Sugarcane bagasse, a byproduct of the cane sugar industry, is an abundant source of hemicellulose that could be hydrolyzed to yield a fermentation feedstock for the production of fuel ethanol and chemicals. The effects of sulfuric acid concentration, temperature, time, and dry matter concentration on hemicellulose hydrolysis were studied with a 20-L batch hydrolysis reactor using a statistical experimental design. Even at less severe conditions considerable amounts (>29%) of the hemicellulose fraction could be extracted. The percentage of soluble oligosaccharides becomes very low in experiments with high yields in monosaccharides, which indicates that the cellulose fraction is only slightly affected. For the sugar yields, acid concentration appears to be the most important parameter, while for the formation of sugar degradation products, temperature shows the highest impact. It could be demonstrated that the dry matter concentration in the reaction slurry has a negative effect on the xylose yield that can be compensated by higher concentrations of sulfuric acid owing to a positive interaction between acid concentration and dry matter contents.

Thermophilic fermentation of hydrolysates: the effect of inhibitors on growth of thermophilic bacteria.

Lignocellulosic biomass has great potential as a cheap feedstock in biological processes to produce biofuels or chemicals; however, dilute acid pretreatment at high temperatures produces undesirable compounds. Toxicity tests were done with inhibitors in standard media, to predict the growth-limiting effects on thermophilic strains. The 22 inhibitors included furfural, levulinic acid, acetic acid, and cinnamaldehyde. Neutralizing reagents and additional treatment steps have been tested.