Acidophilic heterotrophs: basic aspects and technological applications.

Acidophiles comprise a group of microorganisms adapted to live in acidic environments. Despite acidophiles are usually associated with an autotrophic metabolism, more than 80 microorganisms capable of utilizing organic matter have been isolated from natural and man-made environments. The ability to reduce soluble and insoluble iron compounds has been described for many of these species and may be harnessed to develop new or improved mining processes when oxidative bioleaching is ineffective. Similarly, as these microorganisms grow in highly acidic media and the chances of contamination are reduced by the low pH, they may be employed to implement robust fermentation processes. By conducting an extensive literature review, this work presents an updated view of basic aspects and technological applications in biomining, bioremediation, fermentation processes aimed at biopolymers production, microbial electrochemical systems, and the potential use of extremozymes.

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.

High 2,3-butanediol production from glycerol by Raoultella terrigena CECT 4519.

Bioconversion of biodiesel-derived glycerol into 2,3-butanediol has received recently much attention due to its increasing surplus and its multiple uses in industry as bulk chemical. The influence of initial glycerol concentration on 2,3-butanediol production in batch runs has been studied. A concentration higher than 140 g/L produces an inhibitory effect on the final 2,3-butanediol concentration and its production rate. In batch mode, the highest yield respect to the theoretical maximum yield (71%) was reached employing 140 g/L as initial concentration 140 g/L. Based on these results, a high 2,3-butanediol production has been achieved through a fed-batch strategy. The reached 2,3-butanediol concentration was 90.5 g/L from pure glycerol and 80.5 g/L from raw glycerol. The 2,3-butanediol yield respect to the theoretical maximum yield was also improved through the fed-batch operation (90%). To date, this concentration is the highest produced amount employing as biocatalyst a non-pathogenic bacterium (level 1).

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.

Behavior of several pseudomonas putida strains growth under different agitation and oxygen supply conditions.

The growth rate of four strains of Pseudomonas putida, KT2440, KT2442, KTH2, and KTH2 (pESOX3), under different fluid dynamic conditions has been studied. The cultures were conducted in a stirred tank bioreactor by changing the stirrer speed. Several process variables, such as biomass concentration, dissolved oxygen concentration, oxygen mass transfer rate and oxygen uptake rate, have been measured or calculated. Also cell viability was determined by viable colony counting in Petri dishes and culture samples were subjected into a transmission electron microscopy analysis, in order to describe the integrity of the individual cells. The experimental results show that the genetically modified organisms, the strains KTH2 and KTH2 (pESOX3), present a different growth under low agitation conditions, and low oxygen supply level, while the growth of the wild type strains, KT2440 and KT2442, followed the typical sigmoidal evolution that could be described by the logistic equation. The presence of outer membrane vesicles has been observed in the GMO strains. When the cultures were conducted at low stirrer speed, and so at low oxygen transfer rate, these vesicles were detected, indicating the bacterial response to oxidative stress, caused by the catalytic activity of the HpaC enzyme. For all of the strains tested, no hydrodynamic stress has been detected, even at very high agitation levels. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:900-909, 2018.

Study on the effects of several operational variables on the enzymatic batch saccharification of orange solid waste.

In this work, batch enzyme-aided extraction and enzymatic saccharification of blade-milled orange waste was studied. The operation variables for this process were thoroughly analysed. It was determined that batch runs with initial pH values of 5.0 and 5.2 controlled during the first hour, 50°C and 300-500r.p.m. agitation resulted in the best yields, with a limited total and partial first-order enzyme deactivation (for cellulases and polygalacturonidase, respectively). Orange peel waste (OPW) at 6.7% w/w dry solid, 0.22 filter paper units (FPU)/g DS and proportional activities of other enzymes led to over 40g/L free monosaccharides and global yields to glucose over 80%. When using 10.1% w/w DS in these conditions, glucose yield was 63%, with total monosaccharide concentration on top of 50g/L. Similar concentrations were obtained by additional partial drying of OPW to 60% humidity at DS/L ratios near 7.5% (glucose yield >80%).

1,3-Propanediol production from glycerol with a novel biocatalyst Shimwellia blattae ATCC 33430: Operational conditions and kinetics in batch cultivations.

Shimwellia blattae ATCC 33430 as biocatalyst in the conversion of 1,3-propanediol from glycerol is herein evaluated. Several operational conditions in batch cultivations, employing pure and raw glycerol as sole carbon source, were studied. Temperature was studied at shaken bottle scale, while pH control strategy, together with the influence of raw glycerol and its impurities during fermentation were studied employing a 2L STBR. Thereafter, fluid dynamic conditions were considered by changing the stirring speed and the gas supply (air or nitrogen) in the same scale-up experiments. The best results were obtained at a temperature of 37°C, an agitation rate of 200rpm, with free pH evolution from 6.9 and subsequent control at 6.5 and no gas supply during the fermentation, employing an initial concentration of 30g/L of raw glycerol. Under these conditions, the biocatalyst is competitive, leading to results in line with other previous works in the literature in batch conditions, reaching a final concentration of 1,3-propanediol of 13.84g/L, with a yield of 0.45g/g and a productivity of 1.19g/(Lh) from raw glycerol.

1,3-Propanediol production by Klebsiella oxytoca NRRL-B199 from glycerol. Medium composition and operational conditions.

Production of 1,3-propanediol from glycerol using Klebsiella oxytoca NRRL-B199 has been studied. Medium composition has been optimized by means of a statistical design based on the Taguchi method. Strong influences of glycerol and phosphate concentrations have been detected on biomass and product yields. Other factors, such as magnesium concentration and K:Na ratio, have shown a small influence on both responses, biomass and product concentrations. An optimized medium composition has been proposed, leading to a final 1,3-propanediol concentration of 12.4 g/L with a selectivity of 72% with respect to glycerol consumed at shaken bottle-scale. Once the medium composition had been optimized, the scale-up from shaken bottles to STBR was conducted. Several experiments in a 2 L STBR have been conducted in order to determine the best operating conditions concerning temperature and agitation. Under the best operating conditions, i.e., a programmed variable stirring rate ranging from 50 to 100 rpm and a temperature of 37 °C, a final concentration of 13.5 g/L of 1,3-propanediol with a selectivity of 86% with respect to the glycerol consumed was obtained.

Analysis of dibenzothiophene desulfurization in a recombinant Pseudomonas putida strain.

Biodesulfurization was monitored in a recombinant Pseudomonas putida CECT5279 strain. DszB desulfinase activity reached a sharp maximum at the early exponential phase, but it rapidly decreased at later growth phases. A model two-step resting-cell process combining sequentially P. putida cells from the late and early exponential growth phases was designed to significantly increase biodesulfurization.