Metabolic Engineering toward Sustainable Production of Nylon-6.

Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.

Xylose anaerobic conversion by open-mixed cultures.

Xylose is, after glucose, the dominant sugar in agricultural wastes. In anaerobic environments, carbohydrates are converted into volatile fatty acids and alcohols. These can be used as building blocks in biotechnological or chemical processes, e.g., to produce bioplastics. In this study, xylose fermentation by mixed microbial cultures was investigated and compared with glucose under the same conditions. The product spectrum obtained with both substrates was comparable. It was observed that, in the case of xylose, a higher fraction of the carbon was converted into catabolic products (butyrate, acetate, and ethanol) and the biomass yield was approximately 20% lower than on glucose, 0.16 versus 0.21 Cmol X/Cmol S. This lower yield is likely related to the need of an extra ATP during xylose uptake. When submitted to a pulse of glucose, the population cultivated on xylose could instantaneously convert the glucose. No substrate preference was observed when glucose and xylose were fed simultaneously to the continuously operated bioreactor.

Robustness of sludge enriched with short SBR cycles for biological nutrient removal.

In this study, it is proposed that short sequencing batch reactor (SBR) cycles select and maintain a robust and active biomass, able to cope with typical disturbances occurring in wastewater treatment plants. In order to test this hypothesis, an SBR system was subjected to COD, N and P shock loads. It was shown that the sludge enriched in the SBR operated with short cycles was able to rapidly recover from the tested disturbances. COD and N removal recovered within 1-2 days for shock loads of 10 times the standard concentration. The P removal took up to 2-3 sludge ages to fully recover from the COD spike, but the enhanced biological phosphorus removal (EBPR) performance was still able to be totally re-established after each of the tests, even in theoretically adverse conditions for the growth of polyphosphate accumulating organisms.

Diversity of microbial communities in open mixed culture fermentations: impact of the pH and carbon source.

Anaerobic fermentation by an open mixed culture was investigated at different pH values (4-8.5) and with three substrates (glucose, glycerol and xylose). The populations established in each condition were assessed by denaturing gradient gel electrophoresis analysis of the 16S ribosomal RNA gene fragments. The fermentation pattern and the composition of the microbial population were also evaluated when operational variations were imposed (increase of substrate concentration or introduction of a second substrate). The experimental results demonstrated that at low and high pH values, a clearly different fermentation pattern was associated with the dominance of a specialised group of clostridiae. At intermediate pH values, the product spectrum was rather variable and seemed to be sensitive to variations in the microbial community. Different substrates resulted in the establishment of different microbial communities. When fed with a mixture of two substrates, mixotrophic microorganisms (capable of degrading both substrates) were found to overgrow the originally dominant specialists. Overall, the experiments have shown that some operational variables have a clear impact on the fermentation pattern and on the population established. However, a uniform relationship between the process characteristics (associated to a metabolic response) and the microbial population present is not always possible.

Glycerol fermentation by (open) mixed cultures: a chemostat study.

Glycerol is an important byproduct of bioethanol and biodiesel production processes. This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals. Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose. Both reactors operated at a pH of 8 and a dilution rate of 0.1 h(-1). Glycerol was mainly converted into ethanol and formate. When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio. This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations. Glucose fermentation mainly generated acetate, ethanol and butyrate. At higher substrate concentrations, acetate and ethanol were the dominant products. Co-fermentations of glucose-glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol. The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively). The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved.

Influence of the pH on (open) mixed culture fermentation of glucose: a chemostat study.

Catabolic products from anaerobic fermentation processes are potentially of industrial interest. The volatile fatty acids and alcohols produced can be used as building blocks in chemical processes or applied directly as substrates in a mixed culture process to produce bioplastics. Development of such applications requires a predictable and controllable product spectrum of the fermentation process. The aim of the research described in this paper was (i) to investigate the product spectrum of an open mixed culture fermentation (MCF) process as a function of the pH, using glucose as substrate, and (ii) to relate the product spectrum obtained to generalized biochemical and thermodynamic considerations. A chemostat was operated under carbon and energy limitation in order to investigate the pH effect on the product spectrum in a MCF process. A transition from CO(2)/H(2) production at lower pH values to formate production at higher pH values was observed. The ratio of CO(2)/H(2) versus formate production was found to be related to the thermodynamics of formate dehydrogenation to CO(2)/H(2). This transition was associated with a shift in the catabolic products, from butyrate and acetate to ethanol and acetate, likely due to a decrease in the oxidation state of the electron carriers in the cell. The product spectrum of the MCF process as a function of the pH could largely be explained using general biochemical considerations.