Valp1, a Newly Identified Temperate Phage Facilitating Coexistence of Lysogenic and Non-Lysogenic Populations of Vibrio anguillarum.

Vibrio anguillarum is a pathogen for several fish and shellfish species. Its ecology is influenced by diverse factors, including bacteriophages. Here, we identify and characterize a new temperate bacteriophage (Valp1) of V. anguillarum. Valp1 is a myovirus with a 60 nm head and a 90 nm contractile tail. Its double-stranded DNA genome of 42,988 bp contains 68 genes, including a protelomerase gene, typical of telomeric phages. Valp1 inhibits the growth of the virulent strain of V. anguillarum PF4, while the derived lysogenic strain P1.1 presents a slight reduction in its growth but is not affected by the presence of Valp1. Both strains present similar virulence in a larval zebrafish (Danio rerio) model, and only slight differences have been observed in their biochemical profile. Co-culture assays reveal that PF4 and P1.1 can coexist for 10 h in the presence of naturally induced Valp1, with the proportion of PF4 ranging between 28% and 1.6%. By the end of the assay, the phage reached a concentration of ~108 PFU/mL, and all the non-lysogenic PF4 strains were resistant to Valp1. This equilibrium was maintained even after five successive subcultures, suggesting the existence of a coexistence mechanism between the lysogenic and non-lysogenic populations of V. anguillarum in conjunction with the phage Valp1.

Effect of electron donors on CO2 fixation from a model cement industry flue gas by non-photosynthetic microbial communities in batch and continuous reactors.

The aim of this work was to evaluate the effect of different inorganic compounds as electron donors for the capture of CO2 from a model cement flue gas CO2 /O2 /N2 (4.2:13.5:82.3% v/v) using a non-photosynthetic microbial community. The inoculum obtained from a H2 -producing reactor was acclimated to CO2 consumption achieving 100% of CO2 removal after 45 days. Na2 S, MnCl2 , NaNO2 , NH4 Cl, Na2 S2 O3 , and FeCl2 were used as energy source for CO2 fixation by the acclimated microbial community showing different efficiencies, being Na2 S the best electron donor evaluated (100% of CO2 consumption) and FeCl2 the less effective (28% of CO2 consumption). In all treatments, acetate and propionate were the main endpoint metabolites. Moreover, scaling the process to a continuous laboratory biotrickling filter using Na2 S as energy source showed a CO2 consumption of up to 77%. Analysis of the microbial community showed that Na2 S and FeCl2 exerted a strong selection on the microbial members in the community showing significant differences (PERMANOVA, p = 0.0001) compared to the control and the other treatments. Results suggest that the CO2 fixing pathways used by the microbial community in all treatments were the 3-hydroxypropionate-4-hydroxybutyrate cycle and the Wood-Ljungdahl pathway.

Enhancing Phosphorus and Nitrogen Uptake in Maize Crops with Food Industry Biosolids and Azotobacter nigricans.

The problem of phosphorus and nitrogen deficiency in agricultural soils has been solved by adding chemical fertilizers. However, their excessive use and their accumulation have only contributed to environmental contamination. Given the high content of nutrients in biosolids collected from a food industry waste treatment plant, their use as fertilizers was investigated in Zea mays plants grown in sandy loam soil collected from a semi-desert area. These biosolids contained insoluble phosphorus sources; therefore, given the ability of Azotobacter nigricans to solubilize phosphates, this strain was incorporated into the study. In vitro, the suitable conditions for the growth of Z. mays plants were determined by using biosolids as a fertilizer and A. nigricans as a plant-growth-promoting microorganism; in vitro, the ability of A. nigricans to solubilize phosphates, fix nitrogen, and produce indole acetic acid, a phytohormone that promotes root formation, was also evaluated. At the greenhouse stage, the Z. mays plants fertilized with biosolids at concentrations of 15 and 20% (v/w) and inoculated with A. nigricans favored the development of bending strength plants, which was observed on the increased stem diameter (>13.5% compared with the negative control and >7.4% compared with the positive control), as well as a better absorption of phosphorus and nitrogen, the concentration of which increased up to 62.8% when compared with that in the control treatments. The interactions between plants and A. nigricans were observed via scanning electron microscopy. The application of biosolids and A. nigricans in Z. mays plants grown in greenhouses presented better development than when Z. mays plants were treated with a chemical fertilizer. The enhanced plant growth was attributed to the increase in root surface area.

Engineering the Metabolic Landscape of Microorganisms for Lignocellulosic Conversion.

Bacteria and yeast are being intensively used to produce biofuels and high-added-value products by using plant biomass derivatives as substrates. The number of microorganisms available for industrial processes is increasing thanks to biotechnological improvements to enhance their productivity and yield through microbial metabolic engineering and laboratory evolution. This is allowing the traditional industrial processes for biofuel production, which included multiple steps, to be improved through the consolidation of single-step processes, reducing the time of the global process, and increasing the yield and operational conditions in terms of the desired products. Engineered microorganisms are now capable of using feedstocks that they were unable to process before their modification, opening broader possibilities for establishing new markets in places where biomass is available. This review discusses metabolic engineering approaches that have been used to improve the microbial processing of biomass to convert the plant feedstock into fuels. Metabolically engineered microorganisms (MEMs) such as bacteria, yeasts, and microalgae are described, highlighting their performance and the biotechnological tools that were used to modify them. Finally, some examples of patents related to the MEMs are mentioned in order to contextualize their current industrial use.

Bioconversion of H2 and CO2 from dark fermentation to methane: Effect of operating conditions on methane concentration.

The main goal of this study was to assess the methane production in a biotrickling filter (BTF) using a synthetic gas mixture (H2/CO2: 60/40), evaluating the effect of the empty bed gas residence time (EBRT), pH, and temperature. The BTF was inoculated with acclimated granular anaerobic sludge. Three EBRT were tested: 11.6, 5.8, and 2.9 h. The decrease in EBRT (from 11.6 to 5.8 h) increased 1.3-fold the methane content (69 ± 3%) with H2 and CO2 removals of 100% and 24 ± 6%, respectively. The following reduction to 2.9 h showed no effect on CH4 content. The increment of the pH had no significant effect; however, the highest CH4 percentage (74%) was observed at a pH of 8.5. The system showed flexibility to adapt to changes in temperature without drastically diminishing CH4 concentration. In these stages, the principal hydrogenotrophic archaea detected was Methanobacterium flexile. Soluble microbial products such as butanol, caproate, and iso-valerate were detected in all the operating stages. This study demonstrates the potential of methane generation from a dark fermentation gaseous effluent.

Role of xylose from acidic hydrolysates of agave bagasse during biohydrogen production.

This study compares the H2 production from glucose, xylose, and acidic hydrolysates of Agave tequilana bagasse as substrates. The fermentation was performed in a granular sludge reactor operated in two phases: (1) model substrates (glucose and xylose) and (2) acidic hydrolysates at 35 °C, pH 4.5 and a hydraulic retention time of 5.5 h with glucose (10 g L-1) and xylose (12 g L-1). A sequencing batch reactor was used to acclimate the biomass between the glucose and xylose continuous fermentation (with a mixture of xylose-glucose) and acidic hydrolysates. During the discontinuous acclimating step, the xylose/glucose ratio increment negatively affected the H2 productivity. Although the continuous H2 production with xylose was negligible, the co-fermentation with glucose (88-12%) allowed H2 productivity of 2,889 ± 502 mL H2 L-1d-1. An acidic hydrolysate concentration of 3.3 gcarbohydrate L-1 showed a three-fold higher H2 productivity than with a concentration of 10 g L-1. The results indicated that xylose, as the only substrate, was challenging to metabolize by the inoculum, and its mixture with glucose improved the H2 productivity. Therefore, the low H2 productivity with hydrolysates could be related to the presence of xylose.

Simultaneous hydrogen production and decolorization of denim textile wastewater: kinetics of decolorizing of indigo dye by bacterial and fungal strains.

This study proposes the treatment and valorization of denim textile effluents through a fermentative hydrogen production process. Also, the study presents the decolorizing capabilities of bacterial and fungal isolates obtained from the fermented textile effluents. The maximum hydrogen production rate was 0.23 L H2/L-d, achieving at the same time color removal. A total of thirty-five bacteria and one fungal isolate were obtained from the fermented effluents and screened for their abilities to decolorize indigo dye, used as a model molecule. From them, isolates identified as Bacillus BT5, Bacillus BT9, Lactobacillus BT20, Lysinibacillus BT32, and Aspergillus H1T showed notable decolorizing capacities. Lactobacillus BT20 reached 90% of decolorization using glucose as co-substrate after 11 days of incubation producing colorless metabolites. Bacillus BT9 was able to utilize the indigo dye as the sole carbon source achieving a maximum decolorization of 60% after 9 days of incubation and producing a red-colored metabolite. In contrast, Bacillus BT5 and Lysinibacillus BT32 exhibited the lowest percentages of decolorization, barely 33% after 16 and 11 days of incubation, respectively. When Aspergillus H1T was grown in indigo dye supplemented with glucose, 96% of decolorization was reached after 2 days. This study demonstrates the valorization of denim textile effluents for the production of hydrogen via dark fermentation with concomitant color removal.

An ascomycota coculture in batch bioreactor is better than polycultures for cellulase production.

Efficient hydrolysis of holocellulose depends on a proper balance between cellulase (endoglucanase, exoglucanase, ß-glucosidase) and xylanase activities. The present study aimed to induce the production of cellulases and xylanases using liquid cultures (one, two, three, and four fungal strains on the same bioreactor) of wild strains of Trichoderma harzianum, Aspergillus niger, and Fusarium oxysporum. The strains were identified by amplification and analysis of the ITS rDNA region and the obtained sequences were deposited in Genbank. Enzymes (endoglucanase, exoglucansae, ß-glucosidase, and xylanase activities) and the profile of extracellular protein isoforms (SDS-PAGE) produced by different fungal combinations (N = 14) were analyzed by Pearson's correlation matrix and principal component analysis (PCA). According to our results, induction of endoglucanase (19.02%) and ß-glucosidase (6.35%) were obtained after 4 days when A. niger and F. oxysporum were cocultured. The combination of A. niger-T. harzianum produced higher endoglucanase in a shorter time than monocultures. On the contrary, when more than two strains were cultured in the same reactor, the relationships of competition were established, trending to diminish the amount of enzymes and the extracellular protein isoforms produced. The xylanase production was sensible to stress produced by mixed cultures, decreasing their activity. This is important when the aim is to produce cellulase-free xylanase. In addition, exoglucanase activity did not change in the combinations tested.

Biohydrogen, biomethane and bioelectricity as crucial components of biorefinery of organic wastes: a review.

Biohydrogen is a sustainable form of energy as it can be produced from organic waste through fermentation processes involving dark fermentation and photofermentation. Very often biohydrogen is included as a part of biorefinery approaches, which reclaim organic wastes that are abundant sources of renewable and low cost substrate that can be efficiently fermented by microorganisms. The aim of this work was to critically assess selected bioenergy alternatives from organic solid waste, such as biohydrogen and bioelectricity, to evaluate their relative advantages and disadvantages in the context of biorefineries, and finally to indicate the trends for future research and development. Biorefining is the sustainable processing of biomass into a spectrum of marketable products, which means: energy, materials, chemicals, food and feed. Dark fermentation of organic wastes could be the beach-head of complete biorefineries that generate biohydrogen as a first step and could significantly influence the future of solid waste management. Series systems show a better efficiency than one-stage process regarding substrate conversion to hydrogen and bioenergy. The dark fermentation also produces fermented by-products (fatty acids and solvents), so there is an opportunity for further combining with other processes that yield more bioenergy. Photoheterotrophic fermentation is one of them: photosynthetic heterotrophs, such as non-sulfur purple bacteria, can thrive on the simple organic substances produced in dark fermentation and light, to give more H2. Effluents from photoheterotrophic fermentation and digestates can be processed in microbial fuel cells for bioelectricity production and methanogenic digestion for methane generation, thus integrating a diverse block of bioenergies. Several digestates from bioenergies could be used for bioproducts generation, such as cellulolytic enzymes and saccharification processes, leading to ethanol fermentation (another bioenergy), thus completing the inverse cascade. Finally, biohydrogen, biomethane and bioelectricity could contribute to significant improvements for solid organic waste management in agricultural regions, as well as in urban areas.

The influence of total solids content and initial pH on batch biohydrogen production by solid substrate fermentation of agroindustrial wastes.

Hydrogen is a valuable clean energy source, and its production by biological processes is attractive and environmentally sound and friendly. In México 5 million tons/yr of agroindustrial wastes are generated; these residues are rich in fermentable organic matter that can be used for hydrogen production. On the other hand, batch, intermittently vented, solid substrate fermentation of organic waste has attracted interest in the last 10 years. Thus the objective of our work was to determine the effect of initial total solids content and initial pH on H2 production in batch fermentation of a substrate that consisted of a mixture of sugarcane bagasse, pineapple peelings, and waste activated sludge. The experiment was a response surface based on 2(2) factorial with central and axial points with initial TS (15-35%) and initial pH (6.5-7.5) as factors. Fermentation was carried out at 35 °C, with intermittent venting of minireactors and periodic flushing with inert N2 gas. Up to 5 cycles of H2 production were observed; the best treatment in our work showed cumulative H2 productions (ca. 3 mmol H2/gds) with 18% and 6.65 initial TS and pH, respectively. There was a significant effect of TS on production of hydrogen, the latter decreased with initial TS increase from 18% onwards. Cumulative H2 productions achieved in this work were higher than those reported for organic fraction of municipal solid waste (OFMSW) and mixtures of OFMSW and fruit peels waste from fruit juice industry, using the same process. Specific energetic potential due to H2 in our work was attractive and fell in the high side of the range of reported results in the open literature. Batch dark fermentation of agrowastes as practiced in our work could be useful for future biorefineries that generate biohydrogen as a first step and could influence the management of this type of agricultural wastes in México and other countries and regions as well.

Re-fermentation of washed spent solids from batch hydrogenogenic fermentation for additional production of biohydrogen from the organic fraction of municipal solid waste.

In the first batch solid substrate anaerobic hydrogenogenic fermentation with intermittent venting (SSAHF-IV) of the organic fraction of municipal solid waste (OFMSW), a cumulative production of 16.6 mmol H(2)/reactor was obtained. Releases of hydrogen partial pressure first by intermittent venting and afterward by flushing headspace of reactors with inert gas N(2) allowed for further hydrogen production in a second to fourth incubation cycle, with no new inoculum nor substrate nor inhibitor added. After the fourth cycle, no more H(2) could be harvested. Interestingly, accumulated hydrogen in 4 cycles was 100% higher than that produced in the first cycle alone. At the end of incubation, partial pressure of H(2) was near zero whereas high concentrations of organic acids and solvents remained in the spent solids. So, since approximate mass balances indicated that there was still a moderate amount of biodegradable matter in the spent solids we hypothesized that the organic metabolites imposed some kind of inhibition on further fermentation of digestates. Spent solids were washed to eliminate organic metabolites and they were used in a second SSAHF-IV. Two more cycles of H(2) production were obtained, with a cumulative production of ca. 2.4 mmol H(2)/mini-reactor. As a conclusion, washing of spent solids of a previous SSAHF-IV allowed for an increase of hydrogen production by 15% in a second run of SSAHF-IV, leading to the validation of our hypothesis.

Effect of inhibition treatment, type of inocula, and incubation temperature on batch H2 production from organic solid waste.

Two types of induction treatments (heat-shock pretreatment, HSP, and acetylene, Ac), inocula (meso and thermophilic) and incubation temperatures (37 and 55 degrees C) were tested according to a full factorial design 2(3) with the aim of assessing their effects on cumulative H(2) production (P(H), mmol H(2)/mini-reactor), initial H(2) production rate (R(i,H), micromol H(2)/(g VS(i) x h)), lag time (T(lag), h), and metabolites distribution when fermenting organic solid waste with an undefined anerobic consortia in batch mini-reactors. Type of inocula did not have a significant effect on P(H), T(lag), and R(i,H) except for organic acids production: mini-reactors seeded with thermophilic inocula had the highest organic acid production. Concerning the induction treatment, it was found that on the average Ac only affected in a positive way the P(H) and T(lag). Thus, P(H) in Ac-inhibited units (6.97) was 20% larger than those in HSP-inhibited units (5.77). Also, Ac favored a shorter T(lag) for P(H) in comparison with HSP (180 vs. 366). Additionally, a positive correlation was found between H(2) and organic acid production. In contrast, solvent concentration in heat-shocked mini-reactors were slightly higher than in reactors spiked with Ac. Regarding the incubation temperature, on the average mesophilic temperature affected in a positive and very significant way P(H) (10.07 vs. 2.67) and R(i,H) (2.43 vs. 0.76) with minimum T(lag) (87 vs. 459). The positive correlation between H(2) and organic acids production was found again. Yet, incubation temperature did not seem to affect solvent production. A strong interaction was observed between induction treatment and incubation temperature. Thus, Ac-inhibited units showed higher values of P(H) and R(i,H) than that HSP-inhibited units only under thermophilic incubation. Contrary to this, HSP-inhibited units showed the highest values of P(H) and R(i,H) only under mesophilic conditions. Therefore, the superiority of an induction treatment seems to strongly depend on the incubation temperature.

Improvement of biohydrogen production from solid wastes by intermittent venting and gas flushing of batch reactors headspace.

Headspace of batch minireactors was intermittently vented and gas flushed with N2 in order to enhance H2 production (PH) by anaerobic consortia degrading organic solid wastes. Type of inocula (meso and thermophilic), induction treatment (heat-shock pretreatment, HSP, and acetylene, Ac), and incubation temperature (37 and 55 degrees C) were studied by means of a factorial design. On average, it was found that mesophilic incubation had the most significant positive effect on PH followed by treatment with Ac, although the units with the best performance (high values of PH, initial hydrogen production rate, and short lag time) were those HSP-induced units incubated at 37 degrees C (type of inocula was not significant). In this way, after 720 h of incubation PH was inhibited in those units by H2 partial pressure (pH2) of 0.54 atm. Venting and gas flushing with N2 was efficient to eliminate that inhibition achieving additional hydrogen generation in subsequent incubation cycles although smaller than the first one. Thus, four cycles of PH were obtained from the same substrate with neither addition of inocula nor application of induction treatment obtaining an increment of 100% in the generated H2. In those subsequent cycles there was a positive correlation between PH and organic acids/solvent ratio; maximum values were found in the first cycle. Solventogenesis could be clearly distinguished in third and fourth production cycles, probably due to a metabolic shift originated by high organic acid concentrations.