NADH-based kinetic model for acetone-butanol-ethanol production by Clostridium.

We present in this work a kinetic model of the acetone-butanol-ethanol (ABE) fermentation based on enzyme kinetics expressions. The model includes the effect of the co-substrate NADH as a modulating factor of cellular metabolism. The simulations obtained with the model showed an adequate fit to the experimental data reported by several authors, matching or improving the results observed with previous models. In addition, this model does not require artificial mathematical strategies such as on-off functions to achieve a satisfactory fit of the ABE fermentation dynamics. The parametric sensitivity allowed to identify the direct glucose → acetyl-CoA → butyryl-CoA pathway as being more significant for butanol production than the acid re-assimilation pathway. Likewise, model simulations showed that the increase in NADH, due to glucose concentration, favors butanol production and selectivity, finding a maximum selectivity of 3.6, at NADH concentrations above 55 mM and glucose concentration of 126 mM. The introduction of NADH in the model would allow its use for the analysis of electrofermentation processes with Clostridium, since the model establishes a basis for representing changes in the intracellular redox potential from extracellular variables.

Dissimilatory reduction of Fe(III) by a novel Serratia marcescens strain with special insight into the influence of prodigiosin.

A novel pigmented bacterium, initially identified as 11E, was isolated from a site historically known to have various iron-related ores. Phylogenetic analysis of this bacterial strain showed that it belongs to Serratia marcescens. This pigmented S. marcescens 11E cultured individually with glucose, acetate, and glycerol as electron donors along with the soluble electron acceptor iron (Fe) (III) citrate offered a large reduction extent (45.3 %, 31.4 %, and 13.5 %, respectively). On the other hand, when iron oxide (Fe2O3) is used as electron acceptor, the pigmented strain produced a null reduction extent. Surprisingly, the absence of prodigiosin on the bacterial surface (non-pigmented strain) resulted in a large reduction extent of the non-soluble iron form (20-49%). All these extents were comparable and, in some cases, superior to those presented in the literature. Additionally, in the present study, it was found that anthraquinone sulfonate (AQS) stimulated Fe(III) reduction of soluble and non-soluble Fe species only with pigmented S. marcescens. In contrast, in the culture media with the non-pigmented strain, the presence of AQS did not stimulate the Fe(III) reduction. These results suggest that the pigmented phenotype of S. marcescens 11E may perform non-soluble Fe(III) reduction by electron shuttling. In contrast, for the non-pigmented phenotype of this bacterium, non-soluble Fe(III) reduction seems to proceed by direct contact. Our study demonstrates that this bacterium may be used in bioreduction process of heavy metals or as a biocatalyst in bioelectrochemical devices.

Rationale on the High Radical Scavenging Capacity of Betalains.

Betalains are water-soluble natural pigments of increasing importance as antioxidants for pharmaceutical use. Although non-phenolic betalains have lower capacity to scavenge radicals compared to their phenolic analogues, both classes perform well as antioxidants and anti-inflammatory agents in vivo. Here we show that meta-hydroxyphenyl betalain (m-OH-pBeet) and phenylbetalain (pBeet) show higher radical scavenging capacity compared to their N-methyl iminium analogues, in which proton-coupled electron transfer (PCET) from the imine nitrogen atom is precluded. The 1,7-diazaheptamethinium system was found to be essential for the high radical scavenging capacity of betalains and concerted PCET is the most thermodynamically favorable pathway for their one-electron oxidation. The results provide useful insights for the design of nature-derived redox mediators based on the betalain scaffold.

Azo dye biotransformation mediated by AQS immobilized on activated carbon cloth in the presence of microbial inhibitors.

In this work, anthraquinone-2-sulfonate (AQS) was covalently immobilized onto activated carbon cloth (ACC), to be used as redox mediator for the reductive decolorization of reactive red 2 (RR2) by an anaerobic consortium. The immobilization of AQS improved the capacity of ACC to transfer electrons, evidenced by an increment of 3.29-fold in the extent of RR2 decolorization in absence of inhibitors, compared to incubations lacking AQS. Experiments conducted in the presence of vancomycin, an inhibitor of acidogenic bacteria, and with 2-bromoethane sulfonic acid (BES), an inhibitor of methanogenic archaea, revealed that acidogenic bacteria are the main responsible for RR2 biotransformation mediated by immobilized AQS. Nonetheless, the results also suggest that some methanogens are able to maintain their capacity to use immobilized AQS as an electron acceptor to sustain the decolorization process, even in the presence of BES.

A thermostable laccase from Thermus sp. 2.9 and its potential for delignification of Eucalyptus biomass.

Laccases are multicopper oxidases that are being studied for their potential application in pretreatment strategies of lignocellulosic feedstocks for bioethanol production. Here, we report the expression and characterization of a predicted laccase (LAC_2.9) from the thermophilic bacterial strain Thermus sp. 2.9 and investigate its capacity to delignify lignocellulosic biomass. The purified enzyme displayed a blue color typical of laccases, showed strict copper dependence and retained 80% of its activity after 16 h at 70 °C. At 60 °C, the enzyme oxidized 2,2'-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) and 2,6-dimethoxyphenol (DMP) at optimal pH of 5 and 6, respectively. LAC_2.9 had higher substrate specificity (kcat/KM) for DMP with a calculated value that accounts for one of the highest reported for laccases. Further, the enzyme oxidized a phenolic lignin model dimer. The incubation of steam-exploded eucalyptus biomass with LAC_2.9 and 1-hydroxybenzotriazole (HBT) as mediator changed the structural properties of the lignocellulose as evidenced by Fourier transform infrared (FTIR) spectroscopy and thermo-gravimetric analysis (TGA). However, this did not increase the yield of sugars released by enzymatic saccharification. In conclusion, LAC_2.9 is a thermostable laccase with potential application in the delignification of lignocellulosic biomass.

Biotransformation of 4-nitrophenol by co-immobilized Geobacter sulfurreducens and anthraquinone-2-sulfonate in barium alginate beads.

Geobacter sulfurreducens and anthraquinone-2-sulfonate (AQS) were used suspended and immobilized in barium alginate during the biotransformation of 4-nitrophenol (4-NP). The assays were conducted at different concentrations of 4-NP (50-400 mg/L) and AQS, either in suspended (0-400 µM) or immobilized form (0 or 760 µM), and under different pH values (5-9). G. sulfurreducens showed low capacity to reduce 4-NP in absence of AQS, especially at the highest concentrations of the contaminant. AQS improved the reduction rates from 0.0086 h-1, without AQS, to 0.149 h-1 at 400 µM AQS, which represent an increment of 17.3-fold. The co-immobilization of AQS and G. sulfurreducens in barium alginate beads (AQSi-Gi) increased the reduction rates up to 4.8- and 7.2-fold, compared to incubations with G. sulfurreducens in suspended and immobilized form, but in absence of AQS. AQSi-Gi provides to G. sulfurreducens a barrier against the possibly inhibiting effects of 4-NP.

Tailoring partially reduced graphene oxide as redox mediator for enhanced biotransformation of iopromide under methanogenic and sulfate-reducing conditions.

This work reports the first successful application of graphene oxide (GO) and partially reduced GO (rGO) as redox mediator (RM) to increase the biotransformation of the recalcitrant iodinated contrast medium, iopromide (IOP). Results showed that GO-based materials promoted up to 5.5 and 2.8-fold faster biotransformation of IOP by anaerobic sludge under methanogenic and sulfate-reducing conditions, respectively. Correlation between the extent of reduction of GO and its redox-mediating capacity was demonstrated, which was reflected in faster removal and greater extent of biotransformation of IOP. Further analysis indicated that the biotransformation pathway of IOP involved multiple reactions including deiodination, decarboxylation, demethylation, dehydration and N-dealkylation. GO-based materials could be strategically tailored and integrated in biological treatment systems to effectively enhance the redox conversion of recalcitrant pollutants commonly found in wastewater treatment systems and industrial effluents.

Ciprofloxacin toxicity and its co-metabolic removal by a freshwater microalga Chlamydomonas mexicana.

This study evaluated the toxicity and cellular stresses of ciprofloxacin (CIP) and its co-metabolic removal in a freshwater microalga Chlamydomonas mexicana. The toxicological effects of CIP on C. mexicana were assessed by studying the growth and biochemical characteristics of the microalga including total chlorophyll, carotenoid content, malondialdehyde (MDA) and superoxide dismutase (SOD) activity. The calculated effective concentration (EC50) of CIP on C. mexicana was 65±4mgL-1 at 96h. The growth of C. mexicana was significantly inhibited at increased concentrations of CIP, showing 36±1, 75±3. and 88±3% inhibition at 40, 60 and 100mgL-1 CIP, respectively, compared to the control after 11days of cultivation. The total chlorophyll, carotenoid, MDA and SOD activity were significantly increased as a result of relatively high concentrations of CIP stress. C. mexicana showed 13±1% removal of CIP (2mgL-1) after 11days of cultivation; however, the addition of an electron donor (sodium acetate, 4gL-1) highly enhanced the removal of CIP (2mgL-1) by>3-fold after 11days. Kinetic studies showed that removal of CIP followed a first-order model (R2 0.94-0.97) with the apparent rate constants (k) ranging from 0.0121 to 0.079 d-1.

Immobilization of metal-humic acid complexes in anaerobic granular sludge for their application as solid-phase redox mediators in the biotransformation of iopromide in UASB reactors.

Metal-humic acid complexes were synthesized and immobilized by a granulation process in anaerobic sludge for their application as solid-phase redox mediators (RM) in the biotransformation of iopromide. Characterization of Ca- and Fe-humic acid complexes revealed electron accepting capacities of 0.472 and 0.556milli-equivalentsg(-1), respectively. Once immobilized, metal-humic acid complexes significantly increased the biotransformation of iopromide in upflow anaerobic sludge blanket (UASB) reactors. Control UASB reactor (without humic material) achieved 31.6% of iopromide removal, while 80% was removed in UASB reactors supplied with each metal-humic acid complex. Further analyses indicated multiple transformation reactions taking place in iopromide including deiodination, N-dealkylation, decarboxylation and deacetylation. This is the first successful application of immobilized RM, which does not require a supporting material to maintain the solid-phase RM in long term operation of bioreactors. The proposed redox catalyst could be suitable for enhancing the redox conversion of different recalcitrant pollutants present in industrial effluents.

Graphene oxide as electron shuttle for increased redox conversion of contaminants under methanogenic and sulfate-reducing conditions.

Graphene oxide (GO) is reported for the first time as electron shuttle to increase the redox conversion of the azo compound, reactive red 2 (RR2, 0.5mM), and the nitroaromatic, 3-chloronitrobenzene (3CNB, 0.5mM). GO (5mgL(-1)) increased 10-fold and 7.6-fold the reduction rate of RR2 and 3CNB, respectively, in abiotic incubations with sulfide (2.6mM) as electron donor. GO also increased by 2-fold and 3.6-fold, the microbial reduction rate of RR2 by anaerobic sludge under methanogenic and sulfate-reducing conditions, respectively. Deep characterization of GO showed that it has a proper size distribution (predominantly between 450 and 700nm) and redox potential (+50.8mV) to promote the reduction of RR2 and 3CNB. Further analysis revealed that biogenic sulfide plays a major role on the GO-mediated reduction of RR2. GO is proposed as an electron shuttle to accelerate the redox conversion of recalcitrant pollutants, such as nitro-benzenes and azo dyes.

Enhanced microbial decolorization of methyl red with oxidized carbon fiber as redox mediator.

The anaerobic degradation of azo dyes under anaerobic conditions is possible but at a slow rate. Redox mediators (quinones, activated carbon) are used to improve the reduction rate. The aim of this work was to use activated carbon fiber (ACF) as a redox mediator for the anaerobic reduction of the azo dye methyl red. ACF was chemically modified with 8M HNO3 to increase its redox-mediating capacity and used in chemical and anaerobic biological batch assays for the reduction of methyl red. ACF increased its redox-mediating capacity up to 3-fold in chemical assays; in biological assays ACF increased the reduction rate up to 8-fold compared to controls without ACF. However, since the ACF served as support for biomass, a biofilm formed on the fiber significantly reduced its redox-mediating capacity; substrate consumption suggested that the electron transport from ACF to methyl red was the rate-limiting step in the process. These results are the first evidence of the role of ACF as a redox mediator in the reductive decolorization of methyl red, in addition to the effect of biofilm attached to ACF on methyl red reduction. Due to the versatile characteristics of ACF and its redox-mediating capacity, carbon fibers could be used in biological wastewater treatment systems to accelerate the reductive transformation of pollutants commonly found in industrial effluents.