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Screening and bioprospecting of anaerobic consortia for biohydrogen and volatile fatty acid production in a vinasse based medium through dark fermentation
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Screening and bioprospecting of anaerobic consortia for biohydrogen and volatile fatty acid production in a vinasse based medium through dark fermentation ⁎
Eduardo Bittencourt Sydneya, , Alessandra Cristine Novaka, Drielly Rosab, Adriane Bianchi Pedroni Medeirosb, Satinder Kaur Brarc, Christian Larroched, Carlos Ricardo Soccolb Federal University of Technology – Paraná, Department of Bioprocess Engineering and Biotecnology, 84016-210, Ponta Grossa, Paraná, Brazil Federal University of Paraná, Department of Bioprocess Engineering and Biotechnology, Centro Politécnico, 81531-990, Curitiba, Paraná, Brazil Institut National de la Recherche Scientifique, Quebec, Canada d Clermont Université, Université Blaise Pascal, Labex IMobS3, Institut Pascal, Polytech Clermont-Ferrand, 24 avenue des Landais, BP 20206, 63174 Aubière Cedex, France a
b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Anaerobic fermentation Mixed culture Biohydrogen Vinasse Volatile fatty acids
The use of microbial consortia and industrial effluents for the production of biohydrogen by dark fermentation is seen as a key strategy in an attempt to overcome the economic and technical drawbacks of this potential technology. Three mesophilic microbial consortia were sampled and identified. Fermentation was carried out in a vinasse-based medium supplemented with pure or complex carbon sources under different conditions of H2 partial pressure. Consortia LPBAH1 and LPBAH2 were predominantly composed of Oxalobacteraceae and Lactobacillaceae, while LPBAH3 was rich in sporulating Lactobacillaceae (> 96%). Each consortium presented specificities related to biohydrogen and VFAs production: (i) the highest biohydrogen yield was achieved with LPBAH1 (> 50% Oxalobacteraceae) in a vinasse medium supplemented with sugarcane juice (1.59 ± 0.21 molH2/molglucose); (ii) The lower H2 yields were achieved with LPBAH3, which otherwise produced the highest amount of butyric acid (up to 10 g L−1); (iii) LPBAH2 presented great stability in H2 production in different conditions of H2 partial pressure.
1. Introduction The gradual introduction of fuels with increasingly lower carbon content per unit of energy (wood – coal – oil – natural gas) results in a continuous decarbonization of the global fuel mix. This chain of lower carbon content fuel ends in Hydrogen (H2). Currently, the cost of H2 generated from biological processes is very high, especially due to medium cost and process sensitivity. In some bioprocesses for biohydrogen production (such as dark fermentation of organic matter) volatile fatty acids, which are platform molecules that can be used as raw material for green chemistry, are produced and accumulated in the liquid phase. The production and commercialization of these platform molecules will have a great impact on the economics of biohydrogen production technology [1], and thus deserve attention. In the development of biohydrogen processes, two points are of great interest: (i) the use of agroindustry liquid and solid wastes as ⁎
feedstock, promoting economic (and environmental) advantages; and (ii) the use of mixed cultures or consortia. The use of consortia in industrial fermentations presents some challenges, especially related to process stability, but offers less susceptibility to contamination by H2consuming bacteria and oxygen [2]. Moreover, a diverse microbial community is more adaptable to substrate variation (an intrinsic characteristic of (agro)industrial wastewater) due to the presence of alternative metabolic pathways capable of developing a food web where specific groups of organisms maintain a low concentration of critical intermediate products and promote the flux of carbon and electrons from the feedstock material to the desired end product [3]. With respect to the range of potential substrates which can be utilized by the broad range of hydrogen-producing bacteria, it can be stated that, at present, it is a vast and open field for further exploration. One of the major bottlenecks in developing large-scale biohydrogen technologies using waste is its availability and volume of production. Since use of bioH2 is mostly aimed at the production of energy, it can be
Corresponding author. E-mail address: [email protected] (E.B. Sydney).
https://doi.org/10.1016/j.procbio.2018.01.012 Received 3 November 2017; Received in revised form 17 January 2018; Accepted 18 January 2018 1359-5113/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Sydney, E.B., Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.01.012
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considered a fair value-added product, and thus has to be produced in very large quantities. Since the last decade, complex carbon sources, such as molasses [4], food waste [5], dairy wastewater [6], mushroom waste [7], rice slurry [8], cheese whey [9], lignocellulosic materials, glycerol waste [10], vegetable waste [11] and many others have been studied and proved to be viable for the production of biohydrogen by dark fermentation. One of the largest industries in the world is the bioethanol industry. In tropical countries, bioethanol is produced through a classic fermentation process, in which yeasts transform sugarcane juice, molasses, or a molasses-juice mixture into ethanol. At the end of fermentation, almost 100% of the sugar (sucrose) present in the culture medium is consumed by the yeast (usually a Saccharomyces), resulting in a liquid called wine with a yield of approx. 50%. The wine has a concentration of ethanol (% by volume) between 6 and 10° GL, which is recovered by distillation at the top of distillation columns, where the volatile substances are separated based on their different boiling points [12]. From the base of the column, the fermented broth free of ethanol, called vinasse, is removed. Vinasse contains some organic solids in suspension as well as minerals, residual sugar, and some volatile compounds. Considering the ethanol concentration in the wine, vinasse is generated in an average proportion of 12–15 L for each liter of alcohol produced. Because of its production rate and its chemical characteristics (see [13]), vinasse constitutes a large pollution source. It is used indiscriminately as fertilizer, representing a greater risk, especially to soil fertility and underground water [14]. In this perspective, it is of great importance to give a more rational destination to vinasse. In this work, the bioprospecting, screening and identification of microbial consortia capable of producing biohydrogen in the vinassebased medium are presented. Biohydrogen productivity, VFAs production, the effect of partial H2 pressure on the metabolism of the microbial communities and their production stability through time were evaluated.
Table 2 The composition of the vinasse used during the experiments [12]. Parameter
mg/l
Parameter
mg/L
Iron Manganese Lead Cadmium Arsenic pH Nitrate Total Nitrogen (Kjeldahl)
41.8 3.7 < 0.1 < 0.1 < 0.1 4.52 < 10 2.15
Potassium Magnesium Sulfate Total Phosphorus DBO DQO Sodium Calcium
2386 203 1700 104.9 8358 29600 20.1 791
thermocycling, 96 °C were applied for 3 min, 18 cycles of 96 °C for 20 s, 50 °C for 45 s and 68 °C for 1 min. The resulting amplicons were analyzed by electrophoresis in 1.5% agarose gel and quantified with a Qubit kit (Invitrogen). The amplicons were diluted to 16 pM and sequenced on the Illumina MiSeq pallet with the 500V2 set, which generated 250 bp readings. The sequences generated were analyzed using the Qiime program, using a cut line of 16000 readings/sample with the Silva database with 97% identity. 2.3. Medium composition, culture conditions and consortia maintenance Vinasse composition and characterization are described in Table 2. Sucrose (VS), sugarcane juice (VSJ) or molasses (VM) were added in a vinasse medium as a carbon source at an equivalent concentration of 10 g/L of sucrose. Total carbohydrates in molasses were estimated using a refractometer, and in the sugarcane juice by using the Dubois et al. method [16]. The procedure for promoting an anaerobic culture was based on the Balch technique [17]. The removal of oxygen was achieved by boiling the medium under anoxic conditions (CO2 atmosphere). Bicarbonate (1 g/L) was added at 85 °C and Cysteine-HCl at 65 °C as reducing agents to lower the redox potential of the medium. To assure oxygen removal, Resazurin was used as an indicator (0.5 mg/L). The experiments were carried out in 15 mL Hungate tubes, with a working volume of 6 ml, sealed with autoclavable Bakelite screw caps and rubber stoppers, and incubated at 37 °C. The medium pH was adjusted with 1N KOH to 7.0. The cultures were maintained under these conditions for one week and then inoculated in a new medium. Consortia maintenance was carried out by transferring the cell passages to a new fresh medium every two weeks. Monthly production of volatile fatty acids and hydrogen was carried out to evaluate consortia stability.
2. Material and methods 2.1. Consortia sampling Three samples from Brazilian environments with proper conditions for the development of anaerobic microorganisms (known presence of strict/facultative anaerobes and/or absence of oxygen) were collected. The samples were transferred to screw cap glass bottles with the aid of 10 mL sterile syringes (in the case of liquid samples). Their transport was conducted in closed-cell extruded polystyrene foam with dry ice. The name of the strains and the sampling sites are described in Table 1.
2.4. Biohydrogen production and composition analysis 2.2. Molecular identification Biohydrogen production in Hungate tube cultures was periodically measured using 60 ml plastic syringes. Daily gas quantification was carried out in cultures considered free of H2 partial pressure or twice a week (more precisely on the 4th and 7th day of culture) in the cultures where H2 partial pressure was allowed. Gas was collected by inserting a graduated syringe through the flange-type butyl rubber septum and purified for hydrogen content estimation, as discussed later. Purification was carried out by employing CO2 absorption in an NaOH solution [18,19]. The column consisted of a graduated cylinder filled at 50% of its volume with 2 mm glass beads to increase gas contact time with the basic solution. Gas was injected at approximately 3 mL/s through a porous stone.
The microbial identification of consortia LPB AH1, LPB AH2 and LPB AH3 was carried out at the WEMSeq Biotechnology Laboratory (Curitiba/PR – Brazil). For the sequencing and bioinformatics analyses, approximately 5 ml of each consortium sample was taken. The extraction of the total genomic DNA from the samples was performed with phenol/chloroform, followed by the PCR analysis for the V4 region of the 16S rDNA gene with 10 ng of DNA, primers 515F and 806R, in the KlenTaq system (Sigma) according to the methodology of [15]. In Table 1 Origin of the samples collected from the Brazilian environment with potential for biohydrogen and VFAs production. Name
Origin
Location
2.5. Analysis of organic components
LPB AH1 LPB AH2 LPB AH3
Feces from fruit bat (unknown species) Liquid waste from a dairy farm Soil used for sugarcane cultivation
24°35′42.0“S 48°36′17.7“W 25°51′32.9“S 49°29′45.4“W 21°17′13.9“S 47°44′56.1“W
The organic components of the culture medium were determined by using High Performance Liquid Chromatography (HPLC). Before injection, the samples (2 mL) were centrifuged and filtered (Millipore 2
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0.2 μm). The HPLC equipment comprised of Shimadzu Liquid Chromatograph equipped with an Aminex® HPX-87H 300 × 7.8 mm (Bio-Rad) column and a refractive index detector (RID-10A). The column was kept at 60 °C and 5 mM H2SO4 at 0.6 mL/min was used as a mobile phase. The chemical species quantified by this method were glucose, fructose, succinate, lactate, formate, acetate, propionate, and butyrate. All the chemicals were of analytical grade. As the retention time of butyrate and ethanol are very similar, it was impossible to differentiate them with HPLC. The method used for determining ethanol content was based on the oxidation of ethanol to acetic acid by reaction with potassium dichromate in an acidic medium. The solution acquired a green color proportional to the ethanol concentration in the sample, enabling the reading on the spectrophotometer at 600 nm. The standard solution of potassium dichromate (1 L) consisted of the following components: 500 mL of distilled H2O, 325 mL concentrated H2SO4 and 33.678 g of potassium dichromate.
of vertebrates, is described to be related to the excretion of formate [22]. Due to the origin of this consortium (feces of fruit bats) and the presence of formate among the fermentation products O. formigenes is a candidate that plays a key role in this consortium. Despite the representative composition of lactobacillus in the consortium, lactic acid was not detected in the fermented broth. This may be explained by the use of lactic acid as a substrate by other bacteria, resulting in no accumulation at the end of fermentation. LPB AH1 demonstrated a high capacity to produce biohydrogen in a vinasse medium. Higher yield was achieved in VS medium without partial pressure (2.1 molH2/molglucose), statistically different from the yield achieved in VSJ medium (p = .049). This is close to the maximal hydrogen yield expected in mixed cultures [23,24] and corroborates with the observation of [25] that there might be an association between high H2 yields and a low microbial diversity of communities. In the VSJ medium, hydrogen production reached 1.59 ± 0.21 molH2/molglucose, accompanied by considerable amounts of acetate and butyrate production (3.5 and 7.6 g L−1 respectively), which is relevant in coupling with methane or solvent production processes. From Fig. 2, it is observed that in the VSJ medium the effect of H2 partial pressure in H2 production was minimum (yield reduction < 10%, p = .93). This is very interesting considering industrial applications as it facilitates process handling. Fermentation carried out on other complex media (VS and VM) presented relevant negative effects of hydrogen partial pressure. The lower biohydrogen yield in the VM medium can be explained by the inhibitory effect of the sugar components in molasses that influence the microbial growth of bacteria, which can be observed by the lower production of volatile fatty acids in relation to other media (Figs. 2 and 3). On the other hand, the profile of VFAs with and without H2 pressure was very different for the four media (Fig. 2), especially for the VS medium where the composition of VFAs at the end of fermentation was completely altered. The development of acetate consumers in the VM medium was also seen. LPBAH1 presented the potential of acetate consumption probably due to the presence of Archea in the consortia [26] and butyrate production due to the presence of Lactobacilaceae [27].
2.6. Statistical analysis The experimental data of biohydrogen yield obtained from the fermentations of each consortium in different conditions of hydrogen partial pressure and supplemented carbon source was subjected to Student t-tests to determine if the data was significantly different (p < .10). 3. Results and discussion The identification of the microbial composition of consortia demonstrated a similar composition of LPBAH1 and LPBAH2 (approx. 50% of Oxalobacteraceae and 20% Lactobacillaceae). The genus Clostridiaceae was identified in the consortia, LPB AH2 and LPB AH3, while only LPB AH3 presented sporulating Lactobacillaceae (> 96%). 3.1. LPB AH1 The consortium LPB AH1 is composed of the Oxalobacteraceae (52.23% of consortia) family and the genus Lactobacillus (24.29%), while enterobacteria represented only 3% (Fig. 1). Oxalobacteraceae are aerobic/microaerobic, facultative anaerobic and anaerobic bacteria that metabolize oxalate as a major source of carbon for growth [20]. Only a few members of the family are strict anaerobes [21], especially those belonging to the genus Oxalobacter. O. formigenes, for example, a bacterium that colonizes the large intestines
3.2. LPB AH2 Metabolic analysis of the consortium LPB AH2 showed a great potential for biohydrogen production in the vinasse media (Fig. 4), reaching yields between 1.5 and 1.55 molH2/molglucose. No statistical difference of biohydrogen yields was obtained in relation to the carbon source. The effect of hydrogen partial pressure was statistically insignificant for fermentations in sugarcane juice and the sucrose supplemented medium, which is interesting because it facilitates the management of the process on an industrial scale. On the other hand, H2 partial pressure caused a decrease in approx. 100% of the yield in fermentations that used molasses as carbon sources. As observed for LPBAH1, Oxalobacteraceae dominates LPBAH2 composition (Fig. 3), but it led to the production of succinic, lactic and propionic acids. The production of lactic acid in the VSJ medium (Fig. 4) can be explained by the presence of Lactobacillus in the LPB AH2 consortium (Fig. 3). With the H2 partial pressure reduced, this organic acid was not detected, which is in agreement with the expected effects of biohydrogen metabolism (Fig. 4). The presence of succinic and propionic acid can be explained by the presence of lactic acid bacteria but also by the presence of Clostridium strains or other types of bacteria designated as “others”. Lactic acid bacteria were described as producing succinic and propionic acid in fish infusion broth [28], while Clostridium propionicum, for example, is a known and explored producer of propionic acid [29]. The use of molasses as a carbon source together with the maintenance of a low H2 partial pressure environment resulted in the
Fig. 1. Microbial composition of the consortium LPB AH1. The genetic analysis was based on the V4 region of the 16S rDNA gene and classification is presented at family level.
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Fig. 2. Metabolic products of the cultivation of the consortium LPB AH1 in a vinasse medium containing different carbon sources with and without H2 partial pressure; results are the average of 5 analyses and VFAs concentration is shown in g L−1 and biohydrogen (mol H2/mol glucose). Negative results mean consumption of the respective VFA.
reduced products). Pure sucrose was the best carbon source for hydrogen production, with a significantly higher yield than that of sugarcane juice (p = .04 and p = .0005 with and without H2 partial pressure, respectively) (Fig. 5). The use of molasses resulted in a very low hydrogen yield (0.3 and 0.4 molH2/molglucose with and without H2 partial pressure, respectively) but a higher amount of propionic acid in cultures without H2 partial pressure (approx. 14 g/L), which is probably a shift in the microbial community composition caused by molasses that deserves further analysis. The LPBAH3 consortium presented an almost homogeneous composition of Sporolactobacilaceae (96,22%) (Fig. 6). Sporolactobacilaceae resemble Lactobacillus in metabolism, except for the capacity of the first microorganism to respire in the presence of oxygen [30], presenting a variable ability to ferment carbohydrates to lactate and other by-products, such as acetate, ethanol, CO2, formate and succinate [31], which was observed in fermentations carried out with LPBAH3. However, Sporolactobacillaceae are not known to produce propionic acid. The role of Sporolactobacillaceae is poorly described in the literature. Fang et al. [8] and [32] detected Sporolactobacillus in consortia evaluated for hydrogen production but did not discuss its role in the microbial community. Surprisingly, lactic acid was not detected in the fermented medium, which may be caused due to its consumption by bacteria from the Clostridiaceae family [33–36].
Fig. 3. Molecular identification of LPB AH2. The genetic analysis of the microbial community in consortium LPBAH2was based on the V4 region of the 16S rDNA gene and classification is presented at family level.
exclusive production of butyrate in the liquid phase. This showed an interesting role of Lactobacilaceae in the consortium, which is described as a putative butyrate producer both under controlled and non-controlled H2 partial pressure fermentations and/or producing butyrate precursors, such as lactate [27]. 3.3. LPB AH3
3.4. Overall analysis
When cultured in a vinasse medium, the consortium LPB AH3 showed a very high production of butyric acid for all the substrates tested. The highest amount of butyric acid (10 g L−1) among all strains evaluated was produced by this consortium (in a sugarcane juice supplemented medium), but H2 productivity was lowest because considerable amounts of propionate and ethanol were also produced (more
The bioprospecting of biohydrogen and VFAs producing consortia is an interesting tool for the development of high-yield processes since it allows the adaptation of a heterogeneous microbial community in the feedstock. 4
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Fig. 4. Metabolic products of the cultivation of the consortium LPB AH2 in a vinasse medium containing different carbon sources with and without H2 partial pressure; results are the average of 5 analyses and VFAs concentration is shown in g L−1 and biohydrogen (mol H2/mol glucose).
Fig. 5. Metabolic products of the cultivation of the consortium LPB AH3 in a vinasse medium containing different carbon sources with and without H2 partial pressure; results are the average of five analyses and VFAs concentration is shown in g L−1 and biohydrogen (mol H2/mol glucose).
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source and active microbial community play an important role in process development. A previous study [42] using a mesophilic consortium from an anaerobic digester resulted in a higher hydrogen yield of 0.826 molH2/ molglucose (considering 1 g of glucose = 1,07 gCOD) at 20% vinasse (diluted in water – no supplementation of organic carbon). The low hydrogen yield achieved in the referred study is a consequence of a low content of fermentable sugars in vinasse, which reflects the necessity of organic carbon supplementation. Another study [43] achieved a 10 fold increase in hydrogen yield by supplementing vinasse with glucose. A maximum yield of 3.54 molH2/ molglucose was achieved with a mixture of 75% glucose and 25% sugarcane vinasse in a continuous fluidized bed reactor, where bacteria from Prevoltellaceae (55%) and Megasphaera (28%) predominated. The low abundance of the Clostridia bacterium (3%) observed was similar to the LPB AH2 (10%) and LPB AH3 (3%) for this study, although Clostridium is a common microorganism involved in H2 production from complex waste. Different culture operation modes were evaluated by Albanez et al. [44] in a medium composed of 33% molasses + 67% sugarcane vinasse (6 gCOD/l), concluding that a batch mode resulted in higher H2 yields (0.759 molH2/molcarbohydrate) than a fed-batch. It must be considered, however, that sugarcane vinasse is produced in continuous mode and in huge volumes, virtually preventing industrial batch mode operation of hydrogen production processes. Another study [45] describes the use of a continuous operation anaerobic packed bed reactor in biohydrogen production using in natura (pure) sugarcane vinasse, reaching 1.4 molH2/moltotalcarbohydrates but failing in achieving process stability. Biohydrogen yields as high as 1.5–1.6 molH2/molglucose in pure vinasse media supplemented with complex organic carbon sources, achieved in LPBAH1 and LPBAH2, have not been described in the scientific literature to date, which reinforces the enormous potential of LPBAH1 and LPBAH2 for biohydrogen and VFAs production since medium and culture conditions were not optimized.
Fig. 6. Microbial composition of the consortium LPB AH3. Genetic analysis was based on the V4 region of the 16S rDNA gene and classification is presented at family level.
Among the consortia evaluated, LPB AH1 (sampled from feces of fruit bats) and LPB AH2 (sampled from the liquid waste of a dairy farm) showed the highest yields of biohydrogen production and both have higher microbial biodiversity in comparison to LPB AH3, which presented a very homogeneous composition. In terms of VFAs, each of the three consortia presented its own particularities, but all presented at least one condition where platform molecules were produced with interesting profiles and/or quantities. The generation of VFA (amounts and types) was in agreement with the hydrogen yield achieved for each consortium in specific conditions. When comparing hydrogen yields with the literature (usage of complex media), it was observed that the results achieved for consortia LPB AH1 and LPB AH2 lie in the average productivity for mesophilic microorganisms [37], which is 1.46 molH2/molglucose. Distillery effluents are such a potential feedstock due to their chemical characteristics (e.g. Table 2) and volume of production and there are many studies on their use for biohydrogen production [38–41]. However very few studies are focused on the use of sugarcane vinasse. Because the composition of the liquid effluents from distilleries using different raw materials (agave, sugarcane juice, beet, corn) varies greatly, discussion will focus on the microbial community and production of hydrogen specifically from sugarcane vinasse. The data on biohydrogen production from sugarcane vinasse in comparison to the present study is summarized in Table 3. Most studies on sugarcane vinasse bioprocessing for H2 production focus on vinasse concentration and operation mode. Vinasse concentration is described as influencing hydrogen yield in most studies (Table 3), except [42], which showed minimal influence of substrate concentration on microbial community composition (in this case composed of Clostridiaceae, 73%, and Ruminococcaceae, 26%). These indicate that the inoculation
4. Conclusion In this work, the adaptation of three consortia producing biohydrogen and VFAs in an organic carbon supplemented vinasse based medium was successfully achieved. The different behavior of each consortium in the proposed media reinforces the importance of bioprospecting for new consortia and screening new strains for production of biohydrogen and VFAs. In agroindustry based economies, huge volumes of waste is generated and should be reprocessed to avoid environmental issues and promote technological and economic development. Dark fermentation is an alternative to valorize the enormous volume of vinasse that is produced within bioethanol industries in the form of platform molecules and biohydrogen. Knowing the microbial
Table 3 Hydrogen yields from sugarcane vinasse processing achieved by different studies. Inoculum, operation mode and media composition were the operational parameters compared. Inoculum source
operation mode
molH2/molglucose
Medium
Reference
UASB reactor of a poultry slaughterhouse Anaerobic reactor of a poultry slaughterhouse Natural wastewater fermentation Anaerobic sludge for swine manure treatment LPB AH1 LPB AH1
batch batch
0.83 0.76
20% dilluted sugarcane vinasse 33% molasses + 67% sugarcane vinasse
[42] [44]
continuous packed bed reactor continuous fluidized bed reactor batch batch
1.40 3.54
100% sugarcane vinasse 25% sugarcane vinasse + 75% glucose
[45] [43]
2.10 1.59
this study this study
LPB AH2 LPB AH3 LPB AH3
batch batch batch
1.50–1.60 1.15 0.97
100% sucarcane vinasse + 10 g/L sucrose 100% sugarcane vinasse + 10 g/L equivalent sucrose from sugarcane juice 100% sugarcane vinasse + 10 g/L organic carbon sourcea 100% sucarcane vinasse + 10 g/L sucrose 100% sugarcane vinasse + 10 g/L equivalent sucrose from sugarcane juice
a
No statistic difference was observed using sugarcane molasses, sugarcane juice and sucrose.
6
this study this study this study
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community involved is a fundamental step to moving towards overcoming the technical and economic barriers that prevent the application of the technology to the production sector.
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Purohit, P.N. Sarma, Firmicutes with iron dependent hydrogenase drive hydrogen production in anaerobic bioreactor using distillery wastewater, Int. J. Hydrogen Energy 36 (2011) 8234–8242, http://dx.doi.org/10.1016/j.ijhydene. 2011.04.021. [40] G. Buitrón, D. Prato-Garcia, A. Zhang, Biohydrogen production from tequila vinasses using a fixed bed reactor, Water Sci. Technol. 70 (2014) 1919, http://dx.doi. org/10.2166/wst.2014.433. [41] O. García-Depraect, J. Gómez-Romero, E. León-Becerril, A. López-López, A novel biohydrogen production process: co-digestion of vinasse and Nejayote as complex raw substrates using a robust inoculum, Int. J. Hydrogen Energy 42 (2017) 5820–5831, http://dx.doi.org/10.1016/j.ijhydene.2016.11.204. [42] C.Z. Lazaro, V. Perna, C. Etchebehere, M.B.A. Varesche, Sugarcane vinasse as substrate for fermentative hydrogen production: the effects of temperature and substrate concentration, Int. J. Hydrogen Energy 39 (2014) 6407–6418, http://dx. doi.org/10.1016/j.ijhydene.2014.02.058. [43] C.M. dos Reis, M.F. Carosia, I.K. Sakamoto, M.B. Amâncio Varesche, E.L. Silva, Evaluation of hydrogen and methane production from sugarcane vinasse in an anaerobic fluidized bed reactor, Int. J. Hydrogen Energy 40 (2015) 8498–8509, http://dx.doi.org/10.1016/j.ijhydene.2015.04.136. [44] R. Albanez, G. Lovato, M. Zaiat, S.M. Ratusznei, J.A.D. Rodrigues, Optimization, metabolic pathways modeling and scale-up estimative of an AnSBBR applied to biohydrogen production by co-digestion of vinasse and molasses, Int. J. Hydrogen Energy 41 (2016) 20473–20484, http://dx.doi.org/10.1016/j.ijhydene.2016.08. 145. [45] A. Djalma Nunes Ferraz Júnior, J. Wenzel, C. Etchebehere, M. Zaiat, Effect of organic loading rate on hydrogen production from sugarcane vinasse in thermophilic acidogenic packed bed reactors, Int. J. Hydrogen Energy 39 (2014) 16852–16862, http://dx.doi.org/10.1016/j.ijhydene.2014.08.017.
Acknowledgment This work was supported by Fundação Araucária under Grant 18336 and CAPES. References [1] M. de Jong, E.D. Higson, A. Walsh, P. Wellish, IEA Bioenergy–Task 42 Biorefinery. Bio-based Chemicals: Value Added Products from Biorefineries, (2012). [2] M. Jobard, J. Pessiot, R. Nouaille, T. Sime-Ngando, Microbial diversity supporting dark fermentation of waste, Trends Biotechnol. 32 (2014) 549–550, http://dx.doi. org/10.1016/j.tibtech.2014.09.005. [3] B. Angenent, L.T. Wrenn, Optimizing mixed-culture bioprocessing to convert wastes to energy, in: A. Wall, J.D. Harwood, C.S. Deman (Eds.), Bioenergy, ASM Press, 2008, 2018. [4] N. Ren, J. Li, B. Li, Y. Wang, S. Liu, Biohydrogen production from molasses by anaerobic fermentation with a pilot-scale bioreactor system, Int. J. Hydrogen Energy 31 (2006) 2147–2157, http://dx.doi.org/10.1016/j.ijhydene.2006.02.011. [5] P. Agrawal, R. Hema, S. Mahesh Kumar, Experimental investigation on biological hydrogen production using different biomass, J. Teknol. 47 (2007), http://dx.doi. org/10.11113/jt.v47.273. [6] S. Venkata Mohan, V. Lalit Babu, P.N. Sarma, Anaerobic biohydrogen production from dairy wastewater treatment in sequencing batch reactor (AnSBR): effect of organic loading rate, Enzyme Microb. Technol. 41 (2007) 506–515, http://dx.doi. org/10.1016/j.enzmictec.2007.04.007. [7] C.-H. Lay, I.-Y. Sung, G. Kumar, C.-Y. Chu, C.-C. Chen, C.-Y. Lin, Optimizing biohydrogen production from mushroom cultivation waste using anaerobic mixed cultures, Int. J. Hydrogen Energy 37 (2012) 16473–16478, http://dx.doi.org/10. 1016/j.ijhydene.2012.02.135. [8] H. Fang, C. Li, T. Zhang, Acidophilic biohydrogen production from rice slurry, Int. J. Hydrogen Energy 31 (2006) 683–692, http://dx.doi.org/10.1016/j.ijhydene. 2005.07.005. [9] M. Ferchichi, E. Crabbe, G.-H. Gil, W. Hintz, A. Almadidy, Influence of initial pH on hydrogen production from cheese whey, J. Biotechnol. 120 (2005) 402–409, http:// dx.doi.org/10.1016/j.jbiotec.2005.05.017. [10] T. Ito, Y. Nakashimada, K. Senba, T. Matsui, N. Nishio, Hydrogen and ethanol production from glycerol-containing wastes discharged after biodiesel manufacturing process, J. Biosci. Bioeng. 100 (2005) 260–265, http://dx.doi.org/10. 1263/jbb.100.260. [11] S.V. Mohan, G. Mohanakrishna, R.K. Goud, P.N. Sarma, Acidogenic fermentation of vegetable based market waste to harness biohydrogen with simultaneous stabilization, Bioresour. Technol. 100 (2009) 3061–3068, http://dx.doi.org/10.1016/j. biortech.2008.12.059. [12] E.B. Sydney, C.J.D. Neto, A.C. Novak, A.B.P. Medeiros, R. Nouaille, C. Larroche, C.R. Soccol, C.R. Soccol, S.K. Brar, C. Faulds, L.P. Ramos (Eds.), Bioethanol Wastes Economic Valorization, Springer International Publishing, Cham, 2016, pp. 255–289, , http://dx.doi.org/10.1007/978-3-319-30205-8_11. [13] C. Monteiro, Brazilian experience with the disposal of waste water from the cane sugar and alcohol industry, Process Biochem. 10 (1975) 33–41. [14] C.A. Christofoletti, J.P. Escher, J.E. Correia, J.F.U. Marinho, C.S. Fontanetti, Sugarcane vinasse: environmental implications of its use, Waste Manag. 33 (2013) 2752–2761, http://dx.doi.org/10.1016/j.wasman.2013.09.005. [15] J.G. Caporaso, C.L. Lauber, W.A. Walters, D. Berg-Lyons, J. Huntley, N. Fierer, S.M. Owens, J. Betley, L. Fraser, M. Bauer, N. Gormley, J.A. Gilbert, G. Smith, R. Knight, Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms, ISME J. 6 (2012) 1621–1624, http://dx.doi.org/10. 1038/ismej.2012.8. [16] M. DuBois, G. KA, J. Hamilton, F. Rebers, P.A. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. [17] R. Balch, W.E. Wolfe, New approach to the cultivation of methanogenic bacteria: 2mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressurized atmosphere, Appl. Environ. Microbiol. 32 (1976) 781–791. [18] A. Leite, S. Rodrigues, E. Pereira, K. Paulos, A.F. Oliveira, J.M. Lorenzo, A. Teixeira, Physicochemical properties, fatty acid profile and sensory characteristics of sheep and goat meat sausages manufactured with different pork fat levels, Meat Sci. 105 (2015) 114–120, http://dx.doi.org/10.1016/j.meatsci.2015.03.015. [19] M. Mel, M.A.H. Sharuzaman, R.H. Setyobudi, Removal of CO2 from Biogas Plant Using Chemical Absorption Column, (2016), http://dx.doi.org/10.1063/1.4958488 p. 50005. [20] E. Chapelle, R. Mendes, P.A.H. Bakker, J.M. Raaijmakers, Fungal invasion of the rhizosphere microbiome, ISME J. 10 (2016) 265–268, http://dx.doi.org/10.1038/ ismej.2015.82.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Screening and bioprospecting of anaerobic consortia for biohydrogen and volatile fatty acid production in a vinasse based medium through dark fermentation ⁎
Eduardo Bittencourt Sydneya, , Alessandra Cristine Novaka, Drielly Rosab, Adriane Bianchi Pedroni Medeirosb, Satinder Kaur Brarc, Christian Larroched, Carlos Ricardo Soccolb Federal University of Technology – Paraná, Department of Bioprocess Engineering and Biotecnology, 84016-210, Ponta Grossa, Paraná, Brazil Federal University of Paraná, Department of Bioprocess Engineering and Biotechnology, Centro Politécnico, 81531-990, Curitiba, Paraná, Brazil Institut National de la Recherche Scientifique, Quebec, Canada d Clermont Université, Université Blaise Pascal, Labex IMobS3, Institut Pascal, Polytech Clermont-Ferrand, 24 avenue des Landais, BP 20206, 63174 Aubière Cedex, France a
b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Anaerobic fermentation Mixed culture Biohydrogen Vinasse Volatile fatty acids
The use of microbial consortia and industrial effluents for the production of biohydrogen by dark fermentation is seen as a key strategy in an attempt to overcome the economic and technical drawbacks of this potential technology. Three mesophilic microbial consortia were sampled and identified. Fermentation was carried out in a vinasse-based medium supplemented with pure or complex carbon sources under different conditions of H2 partial pressure. Consortia LPBAH1 and LPBAH2 were predominantly composed of Oxalobacteraceae and Lactobacillaceae, while LPBAH3 was rich in sporulating Lactobacillaceae (> 96%). Each consortium presented specificities related to biohydrogen and VFAs production: (i) the highest biohydrogen yield was achieved with LPBAH1 (> 50% Oxalobacteraceae) in a vinasse medium supplemented with sugarcane juice (1.59 ± 0.21 molH2/molglucose); (ii) The lower H2 yields were achieved with LPBAH3, which otherwise produced the highest amount of butyric acid (up to 10 g L−1); (iii) LPBAH2 presented great stability in H2 production in different conditions of H2 partial pressure.
1. Introduction The gradual introduction of fuels with increasingly lower carbon content per unit of energy (wood – coal – oil – natural gas) results in a continuous decarbonization of the global fuel mix. This chain of lower carbon content fuel ends in Hydrogen (H2). Currently, the cost of H2 generated from biological processes is very high, especially due to medium cost and process sensitivity. In some bioprocesses for biohydrogen production (such as dark fermentation of organic matter) volatile fatty acids, which are platform molecules that can be used as raw material for green chemistry, are produced and accumulated in the liquid phase. The production and commercialization of these platform molecules will have a great impact on the economics of biohydrogen production technology [1], and thus deserve attention. In the development of biohydrogen processes, two points are of great interest: (i) the use of agroindustry liquid and solid wastes as ⁎
feedstock, promoting economic (and environmental) advantages; and (ii) the use of mixed cultures or consortia. The use of consortia in industrial fermentations presents some challenges, especially related to process stability, but offers less susceptibility to contamination by H2consuming bacteria and oxygen [2]. Moreover, a diverse microbial community is more adaptable to substrate variation (an intrinsic characteristic of (agro)industrial wastewater) due to the presence of alternative metabolic pathways capable of developing a food web where specific groups of organisms maintain a low concentration of critical intermediate products and promote the flux of carbon and electrons from the feedstock material to the desired end product [3]. With respect to the range of potential substrates which can be utilized by the broad range of hydrogen-producing bacteria, it can be stated that, at present, it is a vast and open field for further exploration. One of the major bottlenecks in developing large-scale biohydrogen technologies using waste is its availability and volume of production. Since use of bioH2 is mostly aimed at the production of energy, it can be
Corresponding author. E-mail address: [email protected] (E.B. Sydney).
https://doi.org/10.1016/j.procbio.2018.01.012 Received 3 November 2017; Received in revised form 17 January 2018; Accepted 18 January 2018 1359-5113/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Sydney, E.B., Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.01.012
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considered a fair value-added product, and thus has to be produced in very large quantities. Since the last decade, complex carbon sources, such as molasses [4], food waste [5], dairy wastewater [6], mushroom waste [7], rice slurry [8], cheese whey [9], lignocellulosic materials, glycerol waste [10], vegetable waste [11] and many others have been studied and proved to be viable for the production of biohydrogen by dark fermentation. One of the largest industries in the world is the bioethanol industry. In tropical countries, bioethanol is produced through a classic fermentation process, in which yeasts transform sugarcane juice, molasses, or a molasses-juice mixture into ethanol. At the end of fermentation, almost 100% of the sugar (sucrose) present in the culture medium is consumed by the yeast (usually a Saccharomyces), resulting in a liquid called wine with a yield of approx. 50%. The wine has a concentration of ethanol (% by volume) between 6 and 10° GL, which is recovered by distillation at the top of distillation columns, where the volatile substances are separated based on their different boiling points [12]. From the base of the column, the fermented broth free of ethanol, called vinasse, is removed. Vinasse contains some organic solids in suspension as well as minerals, residual sugar, and some volatile compounds. Considering the ethanol concentration in the wine, vinasse is generated in an average proportion of 12–15 L for each liter of alcohol produced. Because of its production rate and its chemical characteristics (see [13]), vinasse constitutes a large pollution source. It is used indiscriminately as fertilizer, representing a greater risk, especially to soil fertility and underground water [14]. In this perspective, it is of great importance to give a more rational destination to vinasse. In this work, the bioprospecting, screening and identification of microbial consortia capable of producing biohydrogen in the vinassebased medium are presented. Biohydrogen productivity, VFAs production, the effect of partial H2 pressure on the metabolism of the microbial communities and their production stability through time were evaluated.
Table 2 The composition of the vinasse used during the experiments [12]. Parameter
mg/l
Parameter
mg/L
Iron Manganese Lead Cadmium Arsenic pH Nitrate Total Nitrogen (Kjeldahl)
41.8 3.7 < 0.1 < 0.1 < 0.1 4.52 < 10 2.15
Potassium Magnesium Sulfate Total Phosphorus DBO DQO Sodium Calcium
2386 203 1700 104.9 8358 29600 20.1 791
thermocycling, 96 °C were applied for 3 min, 18 cycles of 96 °C for 20 s, 50 °C for 45 s and 68 °C for 1 min. The resulting amplicons were analyzed by electrophoresis in 1.5% agarose gel and quantified with a Qubit kit (Invitrogen). The amplicons were diluted to 16 pM and sequenced on the Illumina MiSeq pallet with the 500V2 set, which generated 250 bp readings. The sequences generated were analyzed using the Qiime program, using a cut line of 16000 readings/sample with the Silva database with 97% identity. 2.3. Medium composition, culture conditions and consortia maintenance Vinasse composition and characterization are described in Table 2. Sucrose (VS), sugarcane juice (VSJ) or molasses (VM) were added in a vinasse medium as a carbon source at an equivalent concentration of 10 g/L of sucrose. Total carbohydrates in molasses were estimated using a refractometer, and in the sugarcane juice by using the Dubois et al. method [16]. The procedure for promoting an anaerobic culture was based on the Balch technique [17]. The removal of oxygen was achieved by boiling the medium under anoxic conditions (CO2 atmosphere). Bicarbonate (1 g/L) was added at 85 °C and Cysteine-HCl at 65 °C as reducing agents to lower the redox potential of the medium. To assure oxygen removal, Resazurin was used as an indicator (0.5 mg/L). The experiments were carried out in 15 mL Hungate tubes, with a working volume of 6 ml, sealed with autoclavable Bakelite screw caps and rubber stoppers, and incubated at 37 °C. The medium pH was adjusted with 1N KOH to 7.0. The cultures were maintained under these conditions for one week and then inoculated in a new medium. Consortia maintenance was carried out by transferring the cell passages to a new fresh medium every two weeks. Monthly production of volatile fatty acids and hydrogen was carried out to evaluate consortia stability.
2. Material and methods 2.1. Consortia sampling Three samples from Brazilian environments with proper conditions for the development of anaerobic microorganisms (known presence of strict/facultative anaerobes and/or absence of oxygen) were collected. The samples were transferred to screw cap glass bottles with the aid of 10 mL sterile syringes (in the case of liquid samples). Their transport was conducted in closed-cell extruded polystyrene foam with dry ice. The name of the strains and the sampling sites are described in Table 1.
2.4. Biohydrogen production and composition analysis 2.2. Molecular identification Biohydrogen production in Hungate tube cultures was periodically measured using 60 ml plastic syringes. Daily gas quantification was carried out in cultures considered free of H2 partial pressure or twice a week (more precisely on the 4th and 7th day of culture) in the cultures where H2 partial pressure was allowed. Gas was collected by inserting a graduated syringe through the flange-type butyl rubber septum and purified for hydrogen content estimation, as discussed later. Purification was carried out by employing CO2 absorption in an NaOH solution [18,19]. The column consisted of a graduated cylinder filled at 50% of its volume with 2 mm glass beads to increase gas contact time with the basic solution. Gas was injected at approximately 3 mL/s through a porous stone.
The microbial identification of consortia LPB AH1, LPB AH2 and LPB AH3 was carried out at the WEMSeq Biotechnology Laboratory (Curitiba/PR – Brazil). For the sequencing and bioinformatics analyses, approximately 5 ml of each consortium sample was taken. The extraction of the total genomic DNA from the samples was performed with phenol/chloroform, followed by the PCR analysis for the V4 region of the 16S rDNA gene with 10 ng of DNA, primers 515F and 806R, in the KlenTaq system (Sigma) according to the methodology of [15]. In Table 1 Origin of the samples collected from the Brazilian environment with potential for biohydrogen and VFAs production. Name
Origin
Location
2.5. Analysis of organic components
LPB AH1 LPB AH2 LPB AH3
Feces from fruit bat (unknown species) Liquid waste from a dairy farm Soil used for sugarcane cultivation
24°35′42.0“S 48°36′17.7“W 25°51′32.9“S 49°29′45.4“W 21°17′13.9“S 47°44′56.1“W
The organic components of the culture medium were determined by using High Performance Liquid Chromatography (HPLC). Before injection, the samples (2 mL) were centrifuged and filtered (Millipore 2
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0.2 μm). The HPLC equipment comprised of Shimadzu Liquid Chromatograph equipped with an Aminex® HPX-87H 300 × 7.8 mm (Bio-Rad) column and a refractive index detector (RID-10A). The column was kept at 60 °C and 5 mM H2SO4 at 0.6 mL/min was used as a mobile phase. The chemical species quantified by this method were glucose, fructose, succinate, lactate, formate, acetate, propionate, and butyrate. All the chemicals were of analytical grade. As the retention time of butyrate and ethanol are very similar, it was impossible to differentiate them with HPLC. The method used for determining ethanol content was based on the oxidation of ethanol to acetic acid by reaction with potassium dichromate in an acidic medium. The solution acquired a green color proportional to the ethanol concentration in the sample, enabling the reading on the spectrophotometer at 600 nm. The standard solution of potassium dichromate (1 L) consisted of the following components: 500 mL of distilled H2O, 325 mL concentrated H2SO4 and 33.678 g of potassium dichromate.
of vertebrates, is described to be related to the excretion of formate [22]. Due to the origin of this consortium (feces of fruit bats) and the presence of formate among the fermentation products O. formigenes is a candidate that plays a key role in this consortium. Despite the representative composition of lactobacillus in the consortium, lactic acid was not detected in the fermented broth. This may be explained by the use of lactic acid as a substrate by other bacteria, resulting in no accumulation at the end of fermentation. LPB AH1 demonstrated a high capacity to produce biohydrogen in a vinasse medium. Higher yield was achieved in VS medium without partial pressure (2.1 molH2/molglucose), statistically different from the yield achieved in VSJ medium (p = .049). This is close to the maximal hydrogen yield expected in mixed cultures [23,24] and corroborates with the observation of [25] that there might be an association between high H2 yields and a low microbial diversity of communities. In the VSJ medium, hydrogen production reached 1.59 ± 0.21 molH2/molglucose, accompanied by considerable amounts of acetate and butyrate production (3.5 and 7.6 g L−1 respectively), which is relevant in coupling with methane or solvent production processes. From Fig. 2, it is observed that in the VSJ medium the effect of H2 partial pressure in H2 production was minimum (yield reduction < 10%, p = .93). This is very interesting considering industrial applications as it facilitates process handling. Fermentation carried out on other complex media (VS and VM) presented relevant negative effects of hydrogen partial pressure. The lower biohydrogen yield in the VM medium can be explained by the inhibitory effect of the sugar components in molasses that influence the microbial growth of bacteria, which can be observed by the lower production of volatile fatty acids in relation to other media (Figs. 2 and 3). On the other hand, the profile of VFAs with and without H2 pressure was very different for the four media (Fig. 2), especially for the VS medium where the composition of VFAs at the end of fermentation was completely altered. The development of acetate consumers in the VM medium was also seen. LPBAH1 presented the potential of acetate consumption probably due to the presence of Archea in the consortia [26] and butyrate production due to the presence of Lactobacilaceae [27].
2.6. Statistical analysis The experimental data of biohydrogen yield obtained from the fermentations of each consortium in different conditions of hydrogen partial pressure and supplemented carbon source was subjected to Student t-tests to determine if the data was significantly different (p < .10). 3. Results and discussion The identification of the microbial composition of consortia demonstrated a similar composition of LPBAH1 and LPBAH2 (approx. 50% of Oxalobacteraceae and 20% Lactobacillaceae). The genus Clostridiaceae was identified in the consortia, LPB AH2 and LPB AH3, while only LPB AH3 presented sporulating Lactobacillaceae (> 96%). 3.1. LPB AH1 The consortium LPB AH1 is composed of the Oxalobacteraceae (52.23% of consortia) family and the genus Lactobacillus (24.29%), while enterobacteria represented only 3% (Fig. 1). Oxalobacteraceae are aerobic/microaerobic, facultative anaerobic and anaerobic bacteria that metabolize oxalate as a major source of carbon for growth [20]. Only a few members of the family are strict anaerobes [21], especially those belonging to the genus Oxalobacter. O. formigenes, for example, a bacterium that colonizes the large intestines
3.2. LPB AH2 Metabolic analysis of the consortium LPB AH2 showed a great potential for biohydrogen production in the vinasse media (Fig. 4), reaching yields between 1.5 and 1.55 molH2/molglucose. No statistical difference of biohydrogen yields was obtained in relation to the carbon source. The effect of hydrogen partial pressure was statistically insignificant for fermentations in sugarcane juice and the sucrose supplemented medium, which is interesting because it facilitates the management of the process on an industrial scale. On the other hand, H2 partial pressure caused a decrease in approx. 100% of the yield in fermentations that used molasses as carbon sources. As observed for LPBAH1, Oxalobacteraceae dominates LPBAH2 composition (Fig. 3), but it led to the production of succinic, lactic and propionic acids. The production of lactic acid in the VSJ medium (Fig. 4) can be explained by the presence of Lactobacillus in the LPB AH2 consortium (Fig. 3). With the H2 partial pressure reduced, this organic acid was not detected, which is in agreement with the expected effects of biohydrogen metabolism (Fig. 4). The presence of succinic and propionic acid can be explained by the presence of lactic acid bacteria but also by the presence of Clostridium strains or other types of bacteria designated as “others”. Lactic acid bacteria were described as producing succinic and propionic acid in fish infusion broth [28], while Clostridium propionicum, for example, is a known and explored producer of propionic acid [29]. The use of molasses as a carbon source together with the maintenance of a low H2 partial pressure environment resulted in the
Fig. 1. Microbial composition of the consortium LPB AH1. The genetic analysis was based on the V4 region of the 16S rDNA gene and classification is presented at family level.
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Fig. 2. Metabolic products of the cultivation of the consortium LPB AH1 in a vinasse medium containing different carbon sources with and without H2 partial pressure; results are the average of 5 analyses and VFAs concentration is shown in g L−1 and biohydrogen (mol H2/mol glucose). Negative results mean consumption of the respective VFA.
reduced products). Pure sucrose was the best carbon source for hydrogen production, with a significantly higher yield than that of sugarcane juice (p = .04 and p = .0005 with and without H2 partial pressure, respectively) (Fig. 5). The use of molasses resulted in a very low hydrogen yield (0.3 and 0.4 molH2/molglucose with and without H2 partial pressure, respectively) but a higher amount of propionic acid in cultures without H2 partial pressure (approx. 14 g/L), which is probably a shift in the microbial community composition caused by molasses that deserves further analysis. The LPBAH3 consortium presented an almost homogeneous composition of Sporolactobacilaceae (96,22%) (Fig. 6). Sporolactobacilaceae resemble Lactobacillus in metabolism, except for the capacity of the first microorganism to respire in the presence of oxygen [30], presenting a variable ability to ferment carbohydrates to lactate and other by-products, such as acetate, ethanol, CO2, formate and succinate [31], which was observed in fermentations carried out with LPBAH3. However, Sporolactobacillaceae are not known to produce propionic acid. The role of Sporolactobacillaceae is poorly described in the literature. Fang et al. [8] and [32] detected Sporolactobacillus in consortia evaluated for hydrogen production but did not discuss its role in the microbial community. Surprisingly, lactic acid was not detected in the fermented medium, which may be caused due to its consumption by bacteria from the Clostridiaceae family [33–36].
Fig. 3. Molecular identification of LPB AH2. The genetic analysis of the microbial community in consortium LPBAH2was based on the V4 region of the 16S rDNA gene and classification is presented at family level.
exclusive production of butyrate in the liquid phase. This showed an interesting role of Lactobacilaceae in the consortium, which is described as a putative butyrate producer both under controlled and non-controlled H2 partial pressure fermentations and/or producing butyrate precursors, such as lactate [27]. 3.3. LPB AH3
3.4. Overall analysis
When cultured in a vinasse medium, the consortium LPB AH3 showed a very high production of butyric acid for all the substrates tested. The highest amount of butyric acid (10 g L−1) among all strains evaluated was produced by this consortium (in a sugarcane juice supplemented medium), but H2 productivity was lowest because considerable amounts of propionate and ethanol were also produced (more
The bioprospecting of biohydrogen and VFAs producing consortia is an interesting tool for the development of high-yield processes since it allows the adaptation of a heterogeneous microbial community in the feedstock. 4
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Fig. 4. Metabolic products of the cultivation of the consortium LPB AH2 in a vinasse medium containing different carbon sources with and without H2 partial pressure; results are the average of 5 analyses and VFAs concentration is shown in g L−1 and biohydrogen (mol H2/mol glucose).
Fig. 5. Metabolic products of the cultivation of the consortium LPB AH3 in a vinasse medium containing different carbon sources with and without H2 partial pressure; results are the average of five analyses and VFAs concentration is shown in g L−1 and biohydrogen (mol H2/mol glucose).
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source and active microbial community play an important role in process development. A previous study [42] using a mesophilic consortium from an anaerobic digester resulted in a higher hydrogen yield of 0.826 molH2/ molglucose (considering 1 g of glucose = 1,07 gCOD) at 20% vinasse (diluted in water – no supplementation of organic carbon). The low hydrogen yield achieved in the referred study is a consequence of a low content of fermentable sugars in vinasse, which reflects the necessity of organic carbon supplementation. Another study [43] achieved a 10 fold increase in hydrogen yield by supplementing vinasse with glucose. A maximum yield of 3.54 molH2/ molglucose was achieved with a mixture of 75% glucose and 25% sugarcane vinasse in a continuous fluidized bed reactor, where bacteria from Prevoltellaceae (55%) and Megasphaera (28%) predominated. The low abundance of the Clostridia bacterium (3%) observed was similar to the LPB AH2 (10%) and LPB AH3 (3%) for this study, although Clostridium is a common microorganism involved in H2 production from complex waste. Different culture operation modes were evaluated by Albanez et al. [44] in a medium composed of 33% molasses + 67% sugarcane vinasse (6 gCOD/l), concluding that a batch mode resulted in higher H2 yields (0.759 molH2/molcarbohydrate) than a fed-batch. It must be considered, however, that sugarcane vinasse is produced in continuous mode and in huge volumes, virtually preventing industrial batch mode operation of hydrogen production processes. Another study [45] describes the use of a continuous operation anaerobic packed bed reactor in biohydrogen production using in natura (pure) sugarcane vinasse, reaching 1.4 molH2/moltotalcarbohydrates but failing in achieving process stability. Biohydrogen yields as high as 1.5–1.6 molH2/molglucose in pure vinasse media supplemented with complex organic carbon sources, achieved in LPBAH1 and LPBAH2, have not been described in the scientific literature to date, which reinforces the enormous potential of LPBAH1 and LPBAH2 for biohydrogen and VFAs production since medium and culture conditions were not optimized.
Fig. 6. Microbial composition of the consortium LPB AH3. Genetic analysis was based on the V4 region of the 16S rDNA gene and classification is presented at family level.
Among the consortia evaluated, LPB AH1 (sampled from feces of fruit bats) and LPB AH2 (sampled from the liquid waste of a dairy farm) showed the highest yields of biohydrogen production and both have higher microbial biodiversity in comparison to LPB AH3, which presented a very homogeneous composition. In terms of VFAs, each of the three consortia presented its own particularities, but all presented at least one condition where platform molecules were produced with interesting profiles and/or quantities. The generation of VFA (amounts and types) was in agreement with the hydrogen yield achieved for each consortium in specific conditions. When comparing hydrogen yields with the literature (usage of complex media), it was observed that the results achieved for consortia LPB AH1 and LPB AH2 lie in the average productivity for mesophilic microorganisms [37], which is 1.46 molH2/molglucose. Distillery effluents are such a potential feedstock due to their chemical characteristics (e.g. Table 2) and volume of production and there are many studies on their use for biohydrogen production [38–41]. However very few studies are focused on the use of sugarcane vinasse. Because the composition of the liquid effluents from distilleries using different raw materials (agave, sugarcane juice, beet, corn) varies greatly, discussion will focus on the microbial community and production of hydrogen specifically from sugarcane vinasse. The data on biohydrogen production from sugarcane vinasse in comparison to the present study is summarized in Table 3. Most studies on sugarcane vinasse bioprocessing for H2 production focus on vinasse concentration and operation mode. Vinasse concentration is described as influencing hydrogen yield in most studies (Table 3), except [42], which showed minimal influence of substrate concentration on microbial community composition (in this case composed of Clostridiaceae, 73%, and Ruminococcaceae, 26%). These indicate that the inoculation
4. Conclusion In this work, the adaptation of three consortia producing biohydrogen and VFAs in an organic carbon supplemented vinasse based medium was successfully achieved. The different behavior of each consortium in the proposed media reinforces the importance of bioprospecting for new consortia and screening new strains for production of biohydrogen and VFAs. In agroindustry based economies, huge volumes of waste is generated and should be reprocessed to avoid environmental issues and promote technological and economic development. Dark fermentation is an alternative to valorize the enormous volume of vinasse that is produced within bioethanol industries in the form of platform molecules and biohydrogen. Knowing the microbial
Table 3 Hydrogen yields from sugarcane vinasse processing achieved by different studies. Inoculum, operation mode and media composition were the operational parameters compared. Inoculum source
operation mode
molH2/molglucose
Medium
Reference
UASB reactor of a poultry slaughterhouse Anaerobic reactor of a poultry slaughterhouse Natural wastewater fermentation Anaerobic sludge for swine manure treatment LPB AH1 LPB AH1
batch batch
0.83 0.76
20% dilluted sugarcane vinasse 33% molasses + 67% sugarcane vinasse
[42] [44]
continuous packed bed reactor continuous fluidized bed reactor batch batch
1.40 3.54
100% sugarcane vinasse 25% sugarcane vinasse + 75% glucose
[45] [43]
2.10 1.59
this study this study
LPB AH2 LPB AH3 LPB AH3
batch batch batch
1.50–1.60 1.15 0.97
100% sucarcane vinasse + 10 g/L sucrose 100% sugarcane vinasse + 10 g/L equivalent sucrose from sugarcane juice 100% sugarcane vinasse + 10 g/L organic carbon sourcea 100% sucarcane vinasse + 10 g/L sucrose 100% sugarcane vinasse + 10 g/L equivalent sucrose from sugarcane juice
a
No statistic difference was observed using sugarcane molasses, sugarcane juice and sucrose.
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community involved is a fundamental step to moving towards overcoming the technical and economic barriers that prevent the application of the technology to the production sector.
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