New efficient meta-fermentation process for lactic acid production from municipal solid waste.

BACKGROUND: The global market for lactic acid is witnessing growth on the back of increasing applications of lactic acid for manufacturing polylactic acid. Indeed, the lactic acid market is expected to reach 9.8 billion US dollars by 2025. The new concept of meta-fermentation has been proposed in recent years as an alternative to fermentation with pure cultures, due to multiple advantages such as lower susceptibility to contamination, no need for sterilization of culture media or lower raw material costs. However, there are still challenges to overcome to increase the conversion efficiency, decrease formation of by-products and facilitate fermentation control. In this context, the purpose of the study was to develop a robust meta-fermentation process to efficiently produce lactic acid from the OFMSW, stable at pre-industrial scale (1500 L). To maximize lactic acid production, operating conditions (pH, HRT) were modified, and a novel bioaugmentation strategy was tested. RESULTS: A LAB-rich inoculum was generated with LAB isolated from the digestate and grown in the laboratory with MRS medium. After feeding this inoculum to the digester (bioaugmentation), lactic acid accumulation up to 41.5 gO2/L was achieved under optimal operating conditions. This corresponds to more than 70% of the filtered COD measured in the digestate. The amount of lactic acid produced was higher than the volatile fatty acids under all feeding strategies applied. CONCLUSIONS: The operating conditions that enhanced the production of lactic acid from mixed cultures were 55ºC, 2 days HRT and pH 4.8-5.7, with pH-control once a day. The bioaugmentation strategy improved the results obtained in the prototype without applying reinoculation. Lactic acid was the main product along with other carboxylic acids. Further improvements are needed to increase purity as well as lactic acid concentration to reach economic feasibility of the whole process (digestion of OFMSW and downstream).

Identification of the aldolase responsible for the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from natural sterols in Mycolicibacterium smegmatis.

Mycobacterial mutants blocked in ring degradation constructed to achieve C19 synthons production, also accumulate by-products such as C22 intermediates throughout an alternative pathway reducing the production yields and complicating the downstream purification processing of final products. In this work, we have identified the MSMEG_6561 gene, encoding an aldolase responsible for the transformation of 22-hydroxy-3-oxo-cholest-4-ene-24-carboxyl-CoA (22-OH-BCN-CoA) into the 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) precursor (20S)-3-oxopregn-4-ene-20-carboxaldehyde (3-OPA). The deletion of this gene increases the production yield of the C-19 steroidal synthon 4-androstene-3,17-dione (AD) from natural sterols, avoiding the production of 4-HBC as by-product and the drawbacks in the AD purification. The molar yield of AD production using the MS6039-5941-6561 triple mutant strain was checked in flasks and bioreactor improving very significantly compared with the previously described MS6039-5941 strain.

Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one in Mycolicibacterium smegmatis.

The 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) is a C22 steroid synthon of pharmaceutical interest that can be produced as a lateral end-product of the catabolism of natural sterols (e.g., cholesterol or phytosterols). This work studies the role of an aldehyde dehydrogenase coded by the MSMEG_6563 gene of Mycolicibacterium smegmatis, named msRed, in 4-HBC production. This gene is located contiguously to the MSMEG_6561 encoding the aldolase msSal which catalyses the retroaldol elimination of acetyl-CoA of the metabolite intermediate 22-hydroxy-3-oxo-cholest-4-ene-24-carboxyl-CoA to deliver 3-oxo-4-pregnene-20-carboxyl aldehyde (3-OPA). We have demonstrated that msRed reduces 3-OPA to 4-HBC. Moreover, the role of msOpccR reductase encoded by MSMEG_1623 was also explored confirming that it also performs the reduction of 3-OPA into 4-HBC, but less efficiently than msRed. To obtain a M. smegmatis 4-HBC producer strain we deleted MSMEG_5903 (hsd4A) gene in strain MS6039-5941 (ΔkshB1, ΔkstD1) that produces 4-androstene-3,17-dione (AD) from natural sterols (cholesterol or phytosterols). The triple MS6039-5941-5903 mutant was able to produce 9 g/L of 4-HBC from 14 g/L of phytosterols in 2 L bioreactor, showing a productivity of 0.140 g/L h-1. To improve the metabolic flux of sterols towards the production of 4-HBC we have cloned and overexpressed the msSal and msRed enzymes in the MS6039-5941-5903 mutant rendering a production titter of 12.7 g/L with a productivity of 0.185 g/L h-1, and demonstrating that the new recombinant strain has a great potential for its industrial application.

Exploring the potential of slaughterhouse waste valorization: Development and scale-up of a new bioprocess for medium-chain length polyhydroxyalkanoates production.

The progressive increase of slaughterhouse waste production requires actions for both addressing an environmental issue and creating additional value within a biorefinery concept. In this regard, some of these animal by-products exhibit a significant content of fatty acids that could be efficiently converted into bioplastics such as polyhydroxyalkanoates (PHAs) by adequately performing substrate screening with producing bacterial strains and applying affordable pretreatments. One of the main challenges also relies on the difficulty to emulsify these fat-rich substrates within culture broth and make the fatty acids accessible for the producing bacteria. In this work, the potential of two fat-rich animal by-products, grease trap waste (GTW) and tallow-based jelly (TBJ), as inexpensive carbon sources for microbial growth and PHA production was evaluated for the first time. Upon substrate screening, using different pseudomonadal strains (P. resinovorans, P. putida GPo1, P. putida KT2440) and pretreatment conditions (autoclave-based, thermally-treated or saponified substrates), the highest growth and mcl-PHA production performance was obtained for P. resinovorans, thus producing up to 47% w/w mcl-PHA simply using hygienized GTW. The novel bioprocess described in this study was successfully scaled up to 5 and 15 L, resulting in CDW concentrations of 5.9-12.8 g L-1, mcl-PHA contents of 33-62% w/w and PHA yields of 0.1-0.4 gPHA g-1fatty acids, greatly depending on the substrate dosing strategy used and depending on culture conditions. Moreover, process robustness was confirmed along Test Series by the roughly stable monomeric composition of the biopolymer produced, mainly formed by 3-hydroxyoctanoate and 3-hydroxydecanoate. The research here conducted is crucial for the cost-effectiveness of mcl-PHA production along this new slaughterhouse waste-based biorefinery concept.

Modulating redox metabolism to improve isobutanol production in Shimwellia blattae.

BACKGROUND: Isobutanol is a candidate to replace gasoline from fossil resources. This higher alcohol can be produced from sugars using genetically modified microorganisms. Shimwellia blattae (p424IbPSO) is a robust strain resistant to high concentration of isobutanol that can achieve a high production rate of this alcohol. Nevertheless, this strain, like most strains developed for isobutanol production, has some limitations in its metabolic pathway. Isobutanol production under anaerobic conditions leads to a depletion of NADPH, which is necessary for two enzymes in the metabolic pathway. In this work, two independent approaches have been studied to mitigate the co-substrates imbalance: (i) using a NADH-dependent alcohol dehydrogenase to reduce the NADPH dependence of the pathway and (ii) using a transhydrogenase to increase NADPH level. RESULTS: The addition of the NADH-dependent alcohol dehydrogenase from Lactococcus lactis (AdhA) to S. blattae (p424IbPSO) resulted in a 19.3% higher isobutanol production. The recombinant strain S. blattae (p424IbPSO, pIZpntAB) harboring the PntAB transhydrogenase produced 39.0% more isobutanol than the original strain, reaching 5.98 g L-1 of isobutanol. In both strains, we observed a significant decrease in the yields of by-products such as lactic acid or ethanol. CONCLUSIONS: The isobutanol biosynthesis pathway in S. blattae (p424IbPSO) uses the endogenous NADPH-dependent alcohol dehydrogenase YqhD to complete the pathway. The addition of NADH-dependent AdhA leads to a reduction in the consumption of NADPH that is a bottleneck of the pathway. The higher consumption of NADH by AdhA reduces the availability of NADH required for the transformation of pyruvate into lactic acid and ethanol. On the other hand, the expression of PntAB from E. coli increases the availability of NADPH for IlvC and YqhD and at the same time reduces the availability of NADH and thus, the production of lactic acid and ethanol. In this work it is shown how the expression of AdhA and PntAB enzymes in Shimwellia blattae increases yield from 11.9% to 14.4% and 16.4%, respectively.

Dihydroxyacetone production from glycerol using Gluconobacter oxydans: Study of medium composition and operational conditions in shaken flasks.

The production of dihydroxyacetone from glycerol employing aerobic cultures of Gluconobacter oxydans is studied. Dihydroxyacetone is one of the most important value-added products obtained from glycerol, a by-product of biodiesel production. The effect of organic nitrogen source and initial substrate concentrations has been studied together with the possibility of product inhibition. Afterward, the influence of the main operating conditions (temperature, shaking speed, and initial biomass concentration) on in vivo glycerol dehydrogenase activity has also been considered. The results show no evidence of glycerol inhibition, but an important product inhibition was detected, which has been taken into account in a kinetic model for enzymatic activity description. In terms of operating conditions, pH was found to exert a great impact on glycerol conversion, being necessary to keep it above 4 to ensure complete glycerol conversion. The minimum temperature that maximized enzymatic activity was found to be 30°C. In addition, a surprising decoupling between biomass concentration and dihydroxyacetone production rate was observed when adding increasing nitrogen source concentrations at a fixed shaking speed. Glycerol dehydrogenase activity remains constant despite the increase in biomass concentration, contrary to what would be expected. This fact revealed the existence of a rate limiting factor, identified subsequently as oxygen transfer rate depending on the biomass concentration.

Resting cells isobutanol production by Shimwellia blattae (p424IbPSO): Influence of growth culture conditions.

Isobutanol is a promising gasoline additive and could even be a potential substitute used directly as combustible. In this work, the production of isobutanol from glucose by Shimwellia blattae (p424IbPSO) in resting cell cultures is studied. This production has two stages, involving a resting cell phase that has not been studied before. The cell growth was carried out under different operating conditions: temperature and medium composition (YE, ammonium, and IPTG concentrations), looking for the highest isobutanol production. Moreover, the cells were collected at three different growth times checking their isobutanol production capacity. The best operating conditions have been determined as: 30°C of temperature, a medium containing 1.5 g L-1 YE and 1.4 g L-1 of ammonium as nitrogen sources, adding 0.5 mM IPTG as inducer. The cells collected at early growth times are significantly more active. The use of S. blattae (p424IbPSO) in resting cells is a good strategy for the production of isobutanol from glucose yielding better results than in batch growth cultures, a yield of 60% attainment of theoretical maximum yield is obtained under optimal conditions. In addition, it has been demonstrated that if the cells are cultured at higher temperatures and with high IPTG concentrations, inclusion bodies are formed in the cytoplasm inhibiting the isobutanol production in the resting cell stage.