Microbial activity of lactic acid bacteria and hydrogen producers mediated by pH and total solids during the consolidated bioprocessing of agave bagasse.

Lactic acid bacteria (LAB) coexist with Clostridium spp. in hydrogen production processes from complex substrates; however, the role of LAB is still unclear. This study analyzed the fermentation products in a wide range of initial pH (pHi, 5.5-6.9) and total solids (TS%, 8-22%) to determine the activity of these two microbial groups over time (from 24 to 120 h). Agave bagasse served as the feedstock for hydrogen production via consolidated bioprocess (CBP), while the inoculum source was the indigenous mature microbiota. In the early stage of the CBP, hydrogen production from lactic acid occurred only at pHi ≥ 6.0 (ρ = 0.0004) with no effect of TS%; lactic acid accumulated below this pHi value. In this stage, lactic acid production positively correlated with a first cluster of LAB represented by Paucilactobacillus (r = 0.64) and Bacillus (r = 0.81). After 72 h, hydrogen production positively correlated with a second group of LAB led by Enterococcus (r = 0.71) together with the hydrogen producer Clostridium sensu stricto 1 (r = 0.8) and the acetogen Syntrophococcus (r = 0.52) with the influence of TS% (ρ < 0.0001). A further experiment showed that buffering the pH to 6.5 increased and lengthened the lactic acid production, doubling the hydrogen production from 20 to 41 mL H2/gTSadded. This study confirmed the prevalence of distinct groups of LAB over time, whose microbial activity promoted different routes of hydrogen production.

Evaluation of inhibitory compounds produced by bacteria isolated from a hydrogen-producing bioreactor during the self-fermentation of wheat straw.

AIMS: The objective of this study was to evaluate the inhibitory activity of compounds secreted by bacteria isolated from a hydrogen-producing bioreactor to understand how these microorganisms interact in this community. METHODS AND RESULTS: In vitro inhibitory assays were performed using samples secreted by bacteria subject to different treatments to determine if their inhibitory effect was due to organic acids, non-proteinaceous compounds or bacteriocin-like inhibitory substances (BLIS). Bacterial isolated were suppressed 43%, 30% and 27% by neutralized, precipitated and non-neutralized cell-free supernatants, respectively. Non-hydrogen producers (non-H2 P) lactic acid bacteria (LAB) (Lactobacillus plantarum LB1, Lactobacillus pentosus LB7, Pediococcus acidilactici LB4) and hydrogen producers (H2 P) LAB (Enterococcus faecium F) were inhibited by the production of organic acids, non-proteinaceous compounds and BLIS. Meanwhile, the obligate anaerobe H2 P (Clostridium beijerinckii B) inhibited by the production of non-proteinaceous compounds and BLIS. The presence of BLIS was confirmed when proteolytic enzymes affected the inhibitory activity of secreted proteins in values ranging from 20% to 42%. The BLIS produced by L. plantarum LB1, P. acidilactici LB4, L. pentosus LB7 and E. faecium F showed molecular masses of ~11, 25, 20 and 11 kDa, respectively. CONCLUSIONS: It was demonstrated antagonistic interactions between Lactobacillus-Enterococcus and Pediococcus-Enterococcus species, generated by the secretion of organic acids, non-proteinaceous compounds and BLIS. SIGNIFICANCE AND IMPACT OF THE STUDY: We report the interactions between LAB isolated from hydrogen-producing bioreactors. These interactions might impact the dynamics of the microbial population during hydrogen generation. Our work lays a foundation for strategies that allow controlling bacteria that can affect hydrogen production.

The duo Clostridium and Lactobacillus linked to hydrogen production from a lignocellulosic substrate.

The study aimed to identify interspecies interactions within a native microbial community present in a hydrogen-producing bioreactor fed with two wheat straw cultivars. The relationships between the microbial community members were studied building a canonical correspondence analysis and corroborated through in vitro assays. The results showed that the bioreactor reached a stable hydrogen production of ca. 86 mL/kg·d in which the cultivar change did not affect the average performance. Lactobacillus and Clostridium dominated throughout the whole operation period where butyric acid was the main metabolite. A canonical correspondence analysis correlated positively Lactobacillus with hydrogen productivity and hydrogen-producing bacteria like Clostridium and Ruminococaceae. Agar diffusion testing of isolated strains confirmed that Lactobacillus inhibited the growth of Enterococcus, but not of Clostridium. We suggest that the positive interaction between Lactobacillus and Clostridium is generated by a division of labor for degrading the lignocellulosic substrate in which Lactobacillus produces lactic acid from the sugar fermentation while Clostridium quickly uses this lactic acid to produce hydrogen and butyric acid. The significance of this work lies in the fact that different methodological approaches confirm a positive association in the duo Lactobacillus-Clostridium in a bioreactor with stable hydrogen production from a complex substrate.

Effect of transient pH variation on microbial activity and physical characteristics of aerobic granules treating 4-chlorophenol.

Chlorophenols are inhibitory compounds that can be biodegraded by aerobic granules in discontinuous processes. Many industrial wastewaters are characterized by transient pH variation over time. These pH changes could affect the overall granule structure and microbial activity during the chlorophenol biodegradation. The objective of this research was to evaluate the effects of transient pH variation on the specific degradation rate (q), granule integrity coefficient (IC), and size in sequencing batch reactors treating 4-chlorophenol (4-CP). First, aerobic granules were acclimated for efficient 4-CP degradation (>99%). The acclimated granules consisted of 55.7% of the phyla Proteobacteria and 40.6% of Bacteroidetes. The main bacteria belong to the order Sphingobacteriales (24%), as well as Amaricoccus, Acidovorax, Shinella, Rhizobium, and Flavobacterium, some of which are new genera reported in acclimated granules degrading 4-CP. Then, pH changes were applied to the acclimated aerobic granules, observing that acid pHs decreased to a greater extent the specific degradation rate (67% to 99%) than basic pHs (34% to 80%). These pH changes caused the granule disaggregation but with lower effects on the IC. The effects of pH change were mainly on the microbial activity more than the physical characteristics of aerobic granules degrading 4-CP.

Effect of volatile fatty acids mixtures on the simultaneous photofermentative production of hydrogen and polyhydroxybutyrate.

Purple non-sulfur bacteria generate hydrogen and polyhydroxybutyrate (PHB) as a mechanism for disposing of reducing equivalents generated during substrate consumption. However, both pathways compete for the reducing equivalents released from bacteria growing under certain substrates, thus the formation of hydrogen or PHB is detrimental to the formation of each other. The effect of mixtures of acetic, propionic and butyric acids on the formation of H2 and PHB was evaluated using Box-Behnken design. A bacterial community mainly constituted by Rhodopseudomonas palustris was used as inoculum. It was observed that the three volatile fatty acids had a significant effect on the specific PHB production. However, only the propionic acid had a significant effect on the specific H2 production activity and the highest value was observed when acetate was the main component in the mixture. The maximum values for the specific PHB and hydrogen production rates were 16.4 mg-PHB/g-TSS/day and 391 mL-H2/g-TSS/day, respectively.

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.

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.

A review on the factors influencing biohydrogen production from lactate: The key to unlocking enhanced dark fermentative processes.

Dark fermentation (DF) is one of the most promising biological methods to produce bio-hydrogen and other value added bio-products from carbohydrate-rich wastes and wastewater. However, process instability and low hydrogen production yields and rates have been highlighted as the major bottlenecks preventing further development. Numerous studies have associated such concerns with the inhibitory activity of lactate-producing bacteria (LAB) against hydrogen producers. However, an increasing number of studies have also shown lactate-based metabolic pathways as the prevailing platform for hydrogen production. This opens a vast potential to develop new strategies to deal with the "Achilles heel" of DF - LAB overgrowth - while untapping high-performance DF. This review discusses the key factors influencing the lactate-driven hydrogen production, paying particular attention to substrate composition, the operating conditions, as well as the microbiota involved in the process and its potential functionality and related biochemical routes. The current limitations and future perspectives in the field are also presented.

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.

Lactate- and acetate-based biohydrogen production through dark co-fermentation of tequila vinasse and nixtamalization wastewater: Metabolic and microbial community dynamics.

The aim of this work was to study the metabolic and microbial community dynamics during dark co-fermentation of 80% tequila vinasse and 20% nixtamalization wastewater (w/w). Batch co-fermentations were performed in a 3-L well-mixed reactor at 35 °C and pH 5.5. In correspondence to Illumina MiSeq sequencing and reactor monitoring, changes in metabolites and microbial communities were characterized by three main stages: (i) a first stage during which lactate and acetate producers dominated and consumed the major part of fermentable carbohydrates, (ii) a second stage in which lactate and acetate were consumed by emerging hydrogen-producing bacteria (HPB) in correlation with bioH2 (100 NmL/L-h or 1200 NmL/Lreactor) and butyrate production, and (iii) a third stage during which non-HPB outcompeted HPB after bioH2 production ceased. Altogether, the results of this study suggest that cooperative interactions between lactate producers and lactate- and acetate-consuming HPB could be attributed to lactate- and acetate-based cross-feeding interactions.

History of adaptation determines short-term shifts in performance and community structure of hydrogen-producing microbial communities degrading wheat straw.

This study addresses the question of ecological interest for the determination of structure and diversity of microbial communities that degrade lignocellulosic biomasses to produce biofuels. Two microbial consortia with different history, native of wheat straw (NWS) and from a methanogenic digester (MD) fed with cow manure, were contrasted in terms of hydrogen performance, substrate disintegration and microbial diversity. NWS outperformed the hydrogen production rate of MD. Microscopic images revealed that NWS acted on the cuticle and epidermis, generating cellulose strands with high crystallinity, while MD degraded deeper layers, equally affecting all polysaccharides. The bacterial composition markedly differed according to the inocula origin. NWS almost solely comprised hydrogen producers of the phyla Firmicutes and Proteobacteria, with 38% members of Enterococcus. After hydrogen fermentation, NWS comprised 8% Syntrophococcus, an acetogen that cleaves aryl ethers of constituent groups on the aromatic components of lignin. Conversely, MD comprised thirteen phyla, primarily including Firmicutes with H2 -producing members, and Bacteroidetes with non-H2 -producing members, which reduced the hydrogen performance. Overall, the results of this study provide clear evidence that the history of adaptation of NWS enhanced the hydrogen performance from untreated wheat straw. Further, native wheat straw communities have the potential to refine cellulose fibers and produce biofuels simultaneously.

Hydrogen generation via anaerobic fermentation of paper mill wastes.

The objective of this work was to determine the hydrogen production from paper mill wastes using microbial consortia of solid substrate anaerobic digesters. Inocula from mesophilic, continuous solid substrate anaerobic digestion (SSAD) reactors were transferred to small lab scale, batch reactors. Milled paper (used as a surrogate paper waste) was added as substrate and acetylene or 2-bromoethanesulfonate (BES) was spiked for methanogenesis inhibition. In the first phase of experiments it was found that acetylene at 1% v/v in the headspace was as effective as BES in inhibiting methanogenic activity. Hydrogen gas accumulated in the headspace of the bottles, reaching a plateau. Similar final hydrogen concentrations were obtained for reactors spiked with acetylene and BES. In the second phase of tests the headspace of the batch reactors was flushed with nitrogen gas after the first plateau of hydrogen was reached, and subsequently incubated, with no further addition of inhibitor nor substrate. It was found that hydrogen production resumed and reached a second plateau, although somewhat lower than the first one. This procedure was repeated a third time and an additional amount of hydrogen was obtained. The plateaux and initial rates of hydrogen accumulation decreased in each subsequent incubation cycle. The total cumulative hydrogen harvested in the three cycles was much higher (approx. double) than in the first cycle alone. We coined this procedure as IV-SSAH (intermittently vented solid substrate anaerobic hydrogen generation). Our results point out to a feasible strategy for obtaining higher hydrogen yields from the fermentation of industrial solid wastes, and a possible combination of waste treatment processes consisting of a first stage IV-SSAH followed by a second SSAD stage. Useful products of this approach would be hydrogen, organic acids or methane, and anaerobic digestates that could be used as soil amenders after post-treatment.

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.

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.