Poultry slaughterhouse anaerobic ponds as a source of inoculum for biohydrogen production

Journal of Bioscience and Bioengineering VOL. xxx No. xxx, xxx, xxxx www.elsevier.com/locate/jbiosc

Poultry slaughterhouse anaerobic ponds as a source of inoculum for biohydrogen production Fernanda Bernardo Cripa,1, 2, * Mabel Karina Arantes,2 Rodrigo Sequinel,2 Adriana Fiorini,2 Fábio Rogério Rosado,2 and Helton José Alves1, 2 Postgraduate Programme in Bioenergy, Federal University of Paraná UFPR (Sector Palotina), R. Pioneiro, 2153, Jardim Dallas, Palotina, PR 85950-000, Brazil1 and Laboratory of Catalysis and Biofuel Production (LabCatProBio), Federal University of Paraná UFPR (Sector Palotina), R. Pioneiro, 2153, Jardim Dallas, Palotina, PR 85950-000, Brazil2 Received 31 May 2018; accepted 27 July 2019 Available online xxx

Several waste sources have been studied as substrate sources for the production of biogas rich in hydrogen and for the isolation of bacteria capable of fermenting several substrates for the same purpose. Nonetheless, to simplify the process and minimize production costs, it is important to seek alternatives both for the use of microbial consortia using crude waste and for the use of substrates also in their crude form, without the need for purification. The aim of this study was to use only waste as inoculum and substrate for the biological production of hydrogen. Thus, samples from anaerobic ponds of a poultry slaughterhouse were used as inoculum. Sucrose, pure glycerol (in initial tests) and crude glycerol (inserted in blends with pure glycerol) were used as substrates. H2 production experiments were conducted in batches, using a reactor kept in an anaerobic environment for 11 days, at 35
C, under orbital agitation at 150 rpm. To analyse the composition of the biogas and the presence of soluble metabolic products (SMPs), samples of the headspace gases generated and of the reaction medium were collected. The results using sucrose as substrate indicated that the inoculum under study has potential for bio-H2 production, as it produced CH4-free biogas containing 50e60% H2. The inoculum was also shown to be adaptable to the use of glycerine as a substrate, producing biogas with similar characteristics to those obtained from sucrose degradation; however, it required a longer acclimatization period, and thus more in-depth study is required. Ó 2019, The Society for Biotechnology, Japan. All rights reserved. [Key words: Microbial consortia; Glycerol; CH4-free; Biogas; Bioenergy; Residue; Co-product; Anaerobic ponds; Biohydrogen]

Hydrogen (H2) is an important input in processes of the chemical, petrochemical, cosmetic and food industries. Furthermore, it has also been shown to be an interesting alternative as an energetic vector, within the context of searches for new energy sources. Current searches have been guided by economic and environmental factors requiring availability all year round, sustainability and economic viability. H2 can be used in fuel cells and combustion engines without greenhouse gas emissions, due to its high energy capacity of 121,000 kJ kg1 (1). Biological methods, such as fermentative and photosynthetic methods, have been extensively studied for hydrogen production. In this context, dark fermentation is a method that stands out due to its non-dependence on light. Moreover, the hydrogen can be produced from several residual substrates under moderate temperature and pressure conditions (2e4). The use of microbial consortia is one of the alternatives employed to optimize hydrogen production, since complex microbial communities are potentially more tolerant to changes in operating conditions (5,6). These consortia may come from waste treatment systems

* Corresponding author at: Postgraduate Programme in Bioenergy, Federal University of Paraná UFPR (Sector Palotina), R. Pioneiro, 2153, Jardim Dallas, Palotina, PR 85950-000, Brazil. Tel.: þ55 44 3211 8500, þ55 44 9 9920 1410. E-mail address: [email protected] (F.B. Cripa).

such as sludge from wastewater treatment plants or anaerobic reactors, as well as from isolation of media naturally rich in anaerobic microorganisms such as soil and compost. The dark fermentation of organic materials by bacteria presents a promising biohydrogen production path, due to the favourable environment for growth of microorganisms and the high production rate (7,8). Low-value raw materials and high-capacity microorganisms are the two main principles for reducing the cost of biohydrogen production, according to Lay et al. (9). Granular sludge from biodigesters like upflow anaerobic sludge blanket (UASB) are widely used as inoculum in hydrogen production studies (10e13). However, dependence on these biodigesters is a disadvantage in areas where this kind of treatment system is not available, for example, regions that use aerobic and anaerobic ponds as treatment systems. Therefore, this makes it natural to search for alternative sources of anaerobic microbial consortia for H2 production. Some alternatives to the use of granular sludge have been described, such as the use of anaerobic pond suspension applied to the treatment of effluent from the palm oil industry (14) and brewing industry (15), as well as the isolation of microbial consortia from effluent treatment systems (16e18). Rossi et al. (12) using granular sludge from UASB as inoculum and obtained the maximum H2 production of 34%. Palazzi et al. (19) opted to use as inoculum, pure culture of Enterobacter aerogenes

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Please cite this article as: Cripa, F. B et al., Poultry slaughterhouse anaerobic ponds as a source of inoculum for biohydrogen production, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.07.006

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and obtained 37e75% H2 (range of 1.53 mmol H2 mmol glucose1). Already Badiei et al. (14) and Boboescu et al. (15) used anaerobic pond suspension applied to the treatment of effluent from the palm oil industry and brewing industry, respectively, and obtained yield of 50% and 34%. Lin et al. (18), obtained yield of 46% of H2 using sewage sludge as inoculum and observed that the microbial community was composed mainly by four Clostridium species. As shown, the yield using pure culture presents results similar to or higher than the other types of inoculum. However, the interest in the use of the anaerobic pond suspension still has the advantage of not being dependent on sterile conditions throughout the process and less susceptibility to contamination. Characteristics that hinder the use of pure culture for large-scale production. Anaerobic ponds from agroindustry wastewater treatment may be able to offer the intended microbial diversity for mixed H2producing inocula. However, at low concentrations, at least, enrichment and proliferation of the biomass may be required before inoculation for H2 production. Several substrates have been employed in studies of bio-H2 production, which can be both waste, such as wastewater from starch, citrus, dairy (20) and palm oil industries (14), cheese whey (21) and brewery wastewater (22,23), among others, as well as sucrose-based synthetic substrate (24e27). Crude glycerine from biodiesel plants has also been the target of researchers seeking to combine hydrogen production with effluent treatment (10,28,29). It is an easily metabolized substrate, being an interesting raw material also due to the chemical stoichiometry of the reaction involved: one mole of glycerol can produce up to four moles of H2. Crude glycerine corresponds to approximately 10% of the total volume of biodiesel produced (29) and presents a low commercial value as its purity varies greatly depending on the efficiency of the production process or even on the treatment and purification stages (30). In this case, to destine this co-product as a substrate for H2 production, without the need for purification, becomes an interesting alternative both for economic reasons and for the reduction of possible damage to the environment due to its accumulation. Studies using this kind of substrate for H2 production have been reported for different inocula, such as granular sludge from a UASB reactor (10) and pure culture of Bacillus thuringiensis (31), showing satisfactory results with H2 production of 68% and 77%, respectively. The bacterial capacity to produce hydrogen from pure and crude glycerol has been addressed in different studies (32,33). In this way, there is a need to consider an alternative mixed inoculum to granular sludge, and the convenience of using residual glycerine as a substrate for H2 production. The aim of this study was to use only waste as inoculum and substrate for biological production of hydrogen (bio-H2), employing samples from anaerobic ponds applied to the treatment of poultry slaughterhouse wastewater as inoculum, and sucrose, pure glycerol (in initial tests) and crude glycerol (inserted in blends with pure glycerol) as substrate, in order to provide an accessible inoculum with great availability, as well as to contribute to the insertion of glycerol as a substrate in this bioprocess.

Synthetic substrate Synthetic effluent containing 1000 mg L1 of chemical oxygen demand (COD) was prepared as shown in Table 1. The H2 production experiments were conH2 production in batch tests ducted in borosilicate glass flasks with screw caps. The flasks contained a silicone septum for biogas sampling and eventual feed, and were filled with 200 mL of reaction medium, leaving 105 mL of headspace. The H2 production assays were conducted in an anaerobic environment (by N2 flow) at 35
C, orbital agitation at 150 rpm and initial pH of 5.5. The total evaluation period was 7 days. Biogas sampling for compositional analysis was carried out on days 1, 2, and 7. The first assay was considered the control experiment, which had been conducted without the addition of synthetic substrate (100% inoculum), and it was fed with sucrose solution (4 g L1, at 48 h intervals). Tests 2 and 3 were conducted using enriched inoculum and synthetic substrate without reactor feed. In tests 4 and 5, the amount of glycerol inserted in reactor feed was evaluated, using glycerol PA with COD of 200 and 1000 mg O2 L1, respectively. In tests 6 and 7 crude glycerol was introduced into the reactor feed (into blends). Tests 8 and 9 were later planned to eliminate the interference of the crude glycerol observed in tests 6 and 7 in a simple way. For better understanding, the initial characteristics of each test are shown in Table 2. Analytical methods The gases generated in the headspace of the reactor were collected in gasometric ampoules for later analysis by gas chromatography (GC). Analyses were performed using a Thermo Finnigan Trace GC gas chromatograph (Thermo Finnigan Italia S.p.A., Rodano, Milan, Italy) equipped with a thermal conductivity detector (TCD) and a Porapak column (30 m  0.320 mm). The carrier gas used was argon with a flow rate of 2.734 mL min1; injector temperature was 130
C, with flow split into separated flows of 41 mL min1 and an injection volume of 1.0 mL. Oven temperature was set at: initial of 45
C (10 min); final of 130
C (0 min); ramp to 130
C at 15
C min1 starting from 10 min. Analysis of soluble metabolic products (SMPs) was performed by highperformance liquid chromatography (HPLC) in Shimadzu equipment (Shimadzu, Kyoto, Japan), equipped with an LC 20AT pump, a column oven (model CTO-20a), an automatic injector model SIL 20a, UV-Vis detectors (SPD 20a) and an RID 10a refractive index detector, and CBM 20a and Shimadzu LC Solution software. For acid analysis, an Aminex HPx-87H column (300 mm  7.8 mm, Bio-Rad, Hercules, CA, USA) was used, with the following operating conditions: oven temperature was 64
C, the mobile phase consisted of a 0.005% (v/v) sulphuric acid aqueous solution with a flow rate of 0.5 mL, and the UV-Vis detector was set at 210 nm. The same column was used for alcohol analysis, under the following operating conditions: oven temperature was 47
C; the same mobile phase composition was used but with flow of 0.8 mL. To prepare the sample, a 2-mL aliquot was used which was acidified with 80 mL of 2 M sulphuric acid solution. This solution was filtered through a 0.2-mm cellulose acetate membrane.

TABLE 1. Composition of the synthetic substrate. Component

mg L1

Glycerol Urea Potassium phosphate monobasic Potassium phosphate dibasic Sodium phosphate dibasic Calcium chloride dihydrate Nickel nitrate Ferrous sulphate Ferric chloride Cobalt chloride hexahydrate

769.00 11.51 5.36 1.3 2.76 2.06 0.5 2.5 0.25 0.04

Adapted from Fontes Lima and Zaiat (24).

TABLE 2. Initial characteristics of hydrogen production tests. Test

IC (% v/v)

Substrate

Feeding

COD (g O2 L1)

100 20 20 20 20 20 20 20 20

Sucrose Glycerol Glycerol Glycerol Glycerol Glycerol Glycerol Glycerol Glycerol

Sucrose No No Glycerol PA 200 mg O2 L1 Glycerol PA 1000 mg O2 L1 Blend A (3 : 1 v/v) 200 mg O2 L1 Blend A (1 : 1 v/v) 200 mg O2 L1 Blend B (3 : 1 v/v) 200 mg O2 L1 Blend B (1 : 1 v/v) 200 mg O2 L1

13.56 33.5 24.5 34.3 34.3 17.2 17.2 17.2 17.2

Inoculum Samples of watery suspension were collected from anaerobic ponds applied to the treatment of poultry slaughterhouse effluent. They were submitted to thermal pre-treatment (heating in a water bath at 100
C for 1 h) (30) to inactivate the methanogenic bacteria.

1 2 3 4 5 6 7 8 9

Inoculum enrichment Flasks containing the inoculum suspension at pH 5.5 and in an N2 atmosphere were supplemented with sucrose solution (4 g L1) at 48-h intervals for 11 days.

IC, inoculum concentration; COD, chemical oxygen demand; blend A, glycerol PA and crude glycerol without any pre-treatment; blend B, glycerol PA and crude glycerol with pH adjusted to 6.00 and methanol evaporation at 60
C for 15 min.

MATERIALS AND METHODS

Please cite this article as: Cripa, F. B et al., Poultry slaughterhouse anaerobic ponds as a source of inoculum for biohydrogen production, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.07.006

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Determination of COD, volatile solids content and pH was done according to the Standard Methods for the Examination of Water and Wastewater, methods 5220D (34), 2540E (35) and 4500-HþB (36), respectively. Theoretical production of H2 was calculated from chemical reactions considered to convert each substrate in H2, according the general reaction bellow (Eqs. 1 and 2): a substrate / n1 SMP þ b CO2 þ n2 H2

(1)

where a, b, n1 and n2 are the stoichiometric coefficient of each chemical species in the reactions. Theoretical H2 ðmmol H2 =mg SMPsÞ ¼

n2  Csmp  V  1000 n1  MMsmp

3

molasses as substrate. In this study, the maximum percentage of 35.9% H2 in the biogas was obtained using molasses as substrate. Aiming to confirm the results obtained in the control experiment, other tests were performed using inoculum and sucrose feed solution (4 g L1, at 48 h intervals), without the addition of synthetic substrate (100% inoculum). The results showed the production of a high percentage of H2 and the absence of CH4 production (data not shown), confirming the result obtained previously.

(2)

where n1 is stoichiometric coefficient of each SMP, n2 is stoichiometric coefficient of H2 in each equation, Csmp is concentration of SMP generated inside the reactor (mg/ L), V is final volume of reaction medium (L) and MMsmp is molar mass of SMP (mg/ mol). Identification of bacteria present in the inoculum Identification was performed at the beginning of the reaction, using samples of anaerobic pond aqueous suspension pre-treated thermally. Isolation of different colonies was carried out in LuriaeBertani (LB) agar culture medium, for further identification by sequencing. Polymerase chain reaction (PCR) amplification of the 16S rDNA region of the bacteria was performed from a bacterial colony of each isolate, grown in 5 mL of LB liquid medium and held at 28
C for 16 h under agitation (120 rpm). PCR was performed using the forward primer Y1 (50 TGGCTCAGAACGAACGCTGGCGGC-30 ) (37) and the reverse primer Y3 (50 TACCTTGTTACGACTTCACCCCAGTC-30 ) (38), producing an amplicon of approximately 1500 bp. The reaction was performed using 2 mL of culture stock in 1 PCR buffer, 1.5 mM MgCl2, 0.4 mM dNTP mix, 1 unit of Taq DNA Polymerase (Quatro G Pesquisa & Desenvolvimento Ltda, Porto Alegre, RS, Brazil) and 0.2 mM of each primer, in a final volume of 20 mL. It was amplified in a thermal cycler (model MJ 96, Bioer Life Express, Hangzhou, China). The following cycling conditions were used: 94
C for the first 5 min, followed by 30 cycles of 45 s at 94
C, 45 s at 57
C and 45 s at 72
C, and final extension of 5 min at 72
C. Amplification products were analyzed by electrophoresis in 1.5% agarose gel in 1 TBE buffer using a molecular weight standard of 100 pb (Norgen Biotek Corp., Thorold, ON, Canada) containing 0.5 mg mL1 ethidium bromide and photodocumented in L-PIX (Loccus Biotechnology, Cotia, São Paulo, Brazil). For sequencing of the 16S ribosomal gene region, the 1500-bp amplified fragment was eluted from the gel, purified with an EZ-10 elution kit (Bio Basic Inc., Markham, ON, Canada), quantified with a Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA) and sent for sequencing.

RESULTS AND DISCUSSION The main objective of agroindustrial effluent treatment systems is to adapt their physicochemical characteristics to meet the specifications of the current environmental legislation. The liquideffluent treatment process consists of several steps until the effluent is ready to be released into the receiving water body. Anaerobic ponds, which are the source of inoculum studied, generally are the first stage of biological processes. Thus, the concentration of organic matter in the effluents can vary widely, from 1000 to 3700 mg O2 L1 (39), or 1650 mg O2 L1 (40). During H2 production, COD values are slightly altered. It is estimated that the reduction is less than 20%, since most of the organic fraction remains soluble at the end of the fermentative process (41). In the fermentative process, microorganisms use dissolved or suspended organic matter as a substrate, transforming it into gases, water and other value-added chemicals (39). H2 production: sucrose as substrate In the control experiment (Table 1, test 1), using directly the thermally pre-treated bacterial suspension and sucrose as substrate, biogas composed of 58% H2 was obtained after 24 h of reaction, 57% after 48 h, and remaining high until day 7 (37%) (Fig. 1). This evaluation allowed identification of the efficiency of thermal pre-treatment for the inhibition of CH4 production as well as of the balance between consumption rate and production rate of H2 over time. The percentages of H2 obtained in the initial test represent typical biogas of anaerobic fermentation, as described in the literature. Tunçay et al. (42) carried out batch experiments for H2 production. The tests were performed at 35
C and agitation of 125 rpm, using sludge from anaerobic wastewater digesters, sucrose or

H2 production: glycerol as substrate For tests 2 and 3 (Table 2), synthetic substrate containing glycerol at a concentration of 1000 mg O2 L1 was used as a carbon source. In these first tests, H2 production was observed but with a concentration lower than that produced by the sucrose substrate. After only 48 h of reaction, the biogas showed 30% and 16% H2 for tests 2 and 3, respectively. However, on day 7, 63% and 48% H2 was obtained for the tests mentioned, indicating adaptation of the inoculum to the substrate (Fig. 1). A lag phase was observed by Selembo et al. (43), who recorded a minimum lag time of 10 h using glycerol and compost (dewatered sludge) as substrate. The maximum lag time (56 h) was observed using wheat soil as inoculum. On the other hand, hydrogen was not produced when thermally treated digester sludge was used as inoculum. The authors suggested that the inoculum type affects the lag time of the inoculum. Tests were carried out using a higher glycerol concentration, since previous results indicated the ability of the inoculum to metabolize glycerol after a stage of adaptation to the substrate. The glycerol concentration was increased by adding both glycerol PA at concentrations of 200 and 1000 mg O2 L1 (tests 4 and 5) and blends of glycerol PA and crude glycerol at a concentration of 200 mg O2 L1 (tests 6 and 7), results shown in Fig. 2. In test 4, the H2 production observed was only on the seventh day of reaction, being only 5% H2. In test 5, using a higher organic load for feed, the production was even lower (0.4%) and occurred only on the second day of reaction. For tests 6 and 7, in all biogas samples collected, only CO2 was observed. These tests enabled evaluation of the effect of organic loading on inoculum activity. They also showed the ability of the inoculum to degrade the substrate under the initial conditions (1000 mg O2 L1). However, production is inhibited by the addition of more substrate, either glycerol PA or crude glycerol. In all biogas samples collected, only CO2 was observed. For tests 8 and 9, the crude glycerol was pre-treated before mixing it with glycerol PA to ensure that the inhibitory effect was not due to pH and residual methanol. Thus, the pH was adjusted to 6.0 and the residual methanol was eliminated. The results showed that after 48 h of reaction, only in test 9, a significant amount of H2 was observed in the biogas (51%) (Fig. 2). Therefore, the simple and fast residual glycerol pre-treatment strategy (pH adjustment and methanol evaporation) is interesting, it lead to better H2 production. Correlation between volatile organic compounds produced in the reaction medium and metabolic pathways Chromatographic identification of SMPs present in the liquid phase of the reactors (Table 3) allows us to relate the main metabolic pathways established by microorganisms to their respective portions of H2 production. The processes of H2 production are quite complex, especially when microbial consortia are used, since in this case conversions involve more than one metabolic pathway. The stoichiometric equations (Eqs. 3 to 7) indicate the theoretical ratio of H2 and SMPs released by glycerol conversion. On the other hand, Equations 8 and 9 indicate the theoretical ratio of H2 and SMPs released by sucrose conversion.

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Acetic acid C3 H8 O3 þH2 O/CH3 COOH þ CO2 þ3H2

(3)

Acetic acid C12 H22 O11 þ 3H2 O/5CH3 COOH þ 2CO2 þ 4H2

Butyric acid 2C3 H8 O3 / C4 H8 O2 þ 2CO2 þ 4H2

(4)

Butyric acid C12 H22 O11 þ H2 O/2CH3 CH2 CH2 COOH þ 4CO2

Formic acid C3 H8 O3 þ 3H2 O/CH2 O2 þ 2CO2 þ 6H2

(5)

Lactic acid C3 H8 O3 / C3 H6 O3 þ H2

(6)

Propionic acid C3 H8 O3 / C3 H6 O2 þ H2 O

(7)

þ 4H2

(8)

(9)

Fig. 3 shows the conversion of hexoses, pentoses or glycerine to organic acids during the anaerobic fermentation process. All substrates presented (sucrose, glucose, fructose, xylose and glycerol) are processed through the glycolytic pathway to produce H2 (41).

FIG. 1. Concentration of hydrogen as a function of time for tests 1 (substrate sucrose), 2 and 3 (substrate glycerol).

Please cite this article as: Cripa, F. B et al., Poultry slaughterhouse anaerobic ponds as a source of inoculum for biohydrogen production, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.07.006

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FIG. 2. Concentration of hydrogen as a function of time for tests 4 to 9. TABLE 3. Analysis of soluble metabolic products (SMPs) generated during H2 production and theoretical estimated H2 production for each SMP. Acid (mg L1)

Test Sample

HFo HLa HBu 1 1 2 2 3 3 9 9

T0 T7 T0 T2 T0 T7 T0 T2

days days days days

e e 15.4 55.3 0 0 21.8 406.4

e e 0 36 0 14 0 0

86 461 65.9 113.4 0 372.9 58.8 0

Total (mg L1)

HAc

HPr

< bql 842 0 36 0 79.2 28.2 0

< bql < bql 0 36 0 0 30.2 0

Theoretical H2 (mmol H2/mg SMPs) Linked to HFo Linked to HLa Linked to HBu Linked to HAc Linked to HPr

86 1303 81.3 276.7 0 466.1 139.0 406.4

e e 0.40 1.04 e e 0.57 10.03

e e e 0.08 e 0.03 e e

0.39 1.70 0.60 0.43 e 3.39 0.53 e

e 2.24 e 0.36 e 0.79 0.28 e

e e e 0 e e 0 e

Experimental H2 yield (mmol H2 L 1 ) 0 2.4 0 1.2 0 2.3 0 1.00

HFo, formic acid; HLa, lactic acid; HBu, butyric acid; HAc, acetic acid; HPr, propionic acid; < bql, below the quantification limit.

During the oxidative metabolism of glycerol, pyruvate is formed as an intermediate, and it can be used by microorganisms in several ways, resulting in the formation of numerous metabolites (44,45). Using sucrose as substrate, the behaviour of SMPs indicates the adoption of pathways favourable to H2 production, since only butyric and acetic acids were detected at the end of the reaction.

When using glycerol at an initial concentration of 1000 mg O2 L1 as a substrate (tests 2 and 3, which showed H2 production), a predominance of acetic, butyric and formic acids, related to routes with higher H2 production, was observed. However, the presence of lactic and propionic acids, which are related to routes with lower H2 production, was also observed. The presence of lactic acid in reactions

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FIG. 3. Metabolic pathways during the anaerobic fermentation of the following substrates: sucrose, xylose, glucose, fructose and glycerol. Adapted from De Sá et al. (41).

performed with glycerol substrate could be related to the routes of consumption of H2, causing a decrease in its production yield. According to Noike et al. (44), there are two possible explanations involving hydrogen-producing bacteria and lactic acidproducing bacteria: the competition for substrate causing simultaneous production of H2 and lactic acid (observed in the present study); and the inhibition of hydrogen-producing

bacteria due to a substance excreted by lactic acid-producing bacteria. The results obtained for SMP analysis from tests using glycerol as a substrate (tests 5 to 8, which showed unsatisfactory H2 production) did not indicate the presence of any of the SMPs analysed. Therefore, these results were coherent, since no compounds related to H2 production routes were detected.

Please cite this article as: Cripa, F. B et al., Poultry slaughterhouse anaerobic ponds as a source of inoculum for biohydrogen production, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.07.006

VOL. xxx, xxxx Test 9 was the only test using crude glycerol that resulted in H2 production. In the SMP analysis, the presence of formic acid was observed, which is indicative of the route theoretically more favourable to H2 production yield. However, in practice, H2 production was only 1 mmol H2 L1. In general, the results described above show the adoption of pathways favourable for hydrogen production using sucrose as substrate and for the first glycerol condition evaluated. These results corroborate the following argument described by Levin et al. (46): in practice, higher hydrogen yields are associated with the mixture of acetate and butyrate as final fermentation products; on the other hand, the lowest yields are associated with propionate, alcohols and lactic acid, since the conversions involve more than one metabolic pathway. Correlation between H2 production, metabolic pathways and microorganisms present in the microbial consortium The metabolic pathways involved in the reactions are dependent on the microorganisms used, and they directly affect the H2 yield. Therefore, it is important to identify the microorganisms present in the inoculum to understand the results. In the present study, the following three bacterial genera were identified by sequencing of the 16S rDNA gene: Bacillus, Brevibacillus and Paenibacillus. Among these genera, it was possible to identify only Brevibacillus laterosporus at the species level. Molecular confirmation of species of the other genera will be performed later using species-specific primers. Regarding hydrogen production from agroindustrial waste (mixed culture), three classes of microorganisms were found, which are reported by Guo et al. (45) as hydrogen producers, hydrogen consumers and metabolic competitors. Poleto et al. (47) isolated and characterized the bacteria from reactors treating wastewater and activated sludge able to produce biohydrogen from glycerol. As a result, the researchers obtained a great diversity of bacteria capable of growing in medium with glycerol, but not all bacteria had potential for H2 production. Among the diversity of bacteria identified, Bacillus amyloliquefaciens, B. licheniformis, B. subtilis and B. atrophaeus were able to produce H2. On the other hand, Paenibacillus azoreducens and P. cookii did not show this ability. The genus Bacillus is one of the best-known genera of bacteria and it was widely found in the present study, probably due to its ability to withstand adverse temperature conditions. Bacteria of the genus Bacillus are typically rod-shaped, and they produce endospores (48). Bacillus spp. are among the few organisms that have the potential to metabolize glycerol to produce energy and biopolymers (32). Several researchers refer to Bacillus sp. as potential H2 producers, and use it as inoculum in their studies (49,50). Bacillus sp. has also been found in H2-producing reactors after thermal treatment due to its ability to form spores (51). However, some bacteria of the genus Bacillus have also been mentioned as substrate competitors or H2 production inhibitors; one example is lactic acidproducing bacteria (52). Besides the genus Bacillus, the genera Brevibacillus and Paenibacillus are recognized for their biotechnological potential as producers of enzymes, such as proteases, cellulases and amylases, which are capable of degrading organic matter (53). Despite the wide industrial use of bacteria of the genus Paenibacillus and studies of its genome, not much is known about its operational metabolic pathways (54), or about the biochemical and morphological characteristics and aptitude of the genera Brevibacillus and Paenibacillus for H2 production. Hydrogen-consuming bacteria are also a problem to be considered. Thus, the low H2 yield obtained in the present study can be attributed to the homoacetogenesis that is performed by autotrophic microorganisms. Under stress conditions or after

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substrate depletion, autotrophic microorganisms consume H2 and CO2 for acetate production. The difference between acetogenic and homoacetogenic bacteria is a trophic or metabolic condition (52). Therefore, it is important to find ways to control homoacetogenesis and, at the same time, to benefit acidogenesis. Although thermal pre-treatment is capable of preventing methanogenesis, it may not have prevented homoacetogenesis. According to Oh et al. (55), thermal treatment may not be successful in the elimination of homoacetogenic microorganisms. Saady (52) also emphasized that no method of methanogenesis inhibition is successful in eliminating these microorganisms. This is associated with the similarity of most of the characteristics of hydrogen-producing and homoacetogenic bacteria (such as spore formation and the same optimum pH range). Studying mixed culture obtained from an ethanol manufacturing plant and a municipal drinking-water treatment plant, Pendyala et al. (56) identified Clostridium sp., Bacillus sp. and Enterococcus sp. as H2 producers; and Propionibacterium acnes, Brevibacillus sp. and Bacteroides fragilis as homoacetogenics and H2 consumers. In this way, in the present study, H2 production was attributed only to bacteria of the genus Bacillus. This study identified that inoculum coming from anaerobic ponds applied to the treatment of thermally pre-treated poultry slaughterhouse wastewater has potential for bio-H2 production, generating only CO2 as a gaseous co-product of the reaction. The inoculum was also shown to be adaptable to the substrate glycerol. However, the non-use of adaptation steps for substrate exchange may have contributed to the low H2 production yield when using glycerol. It suggests the need for more in-depth studies regarding adaptation of inoculum to the substrate and optimization of the reaction. The test with crude glycerol addition, which presented a favourable route for H2 production, was test 9. It is recommended to conduct further studies on H2 production for the same inoculum and substrate using the conditions of test 9 as a starting point. ACKNOWLEDGMENTS The authors thank the Higher Education Personnel Improvement Coordination (CAPES) for granting scholarship. The authors declare no conflict of interest. References 1. Alves, H. J., Bley Junior, C., Niklevics, R. R., Frigo, E. P., Frio, M. S., and Coimbra-Araújo, C. H.: Overview of hydrogen production technologies from biogas and the applications in fuel cells, Int. J. Hydrogen Energy, 38, 5215e5225 (2013). 2. Junghare, M., Subudhi, S., and Lal, B.: Improvement of hydrogen production under decreased partial pressure by newly isolated alkaline tolerant anaerobe, Clostridium butyricum TM-9A: optimization of process parameters, Int. J. Hydrogen Energy, 37, 3160e3168 (2012). 3. Kadier, A., Kalil, M. S., Abdeshahian, P., Chandrasekhar, K., Mohamed, A., Azman, N. F., Logroño, W., Simayi, Y., and Hamid, A. A.: Recent advances and emerging challenges in microbial electrolysis cells (MECs) for microbial production of hydrogen and value-added chemicals, Renew. Sust. Energ. Rev., 61, 501e525 (2016). 4. Ghimire, A., Frunzo, L., Pirozzi, F., Trably, E., Escudie, R., Lens, P. N. L., and Esposito, G.: A review on dark fermentative biohydrogen production from organic biomass: process parameters and use of by-products, Appl. Energy, 144, 73e95 (2015). 5. Hallenbeck, P. C. and Ghosh, D.: Advances in fermentative biohydrogen production: the way forward? Trends Biotechnol., 27, 287e297 (2009). 6. Hung, C. H., Chang, Y. T., and Chang, Y. J.: Roles of microorganisms other than Clostridium and Enterobacter in anaerobic fermentative biohydrogen production systems - a review, Bioresour. Technol., 102, 8437e8444 (2011). 7. Ren, N., Zhao, L., Chen, C., Guo, W., and Cao, G.: A review on bioconversion of lignocellulosic biomass to H2: key challenges and new insights, Bioresour. Technol., 215, 92e99 (2016).

Please cite this article as: Cripa, F. B et al., Poultry slaughterhouse anaerobic ponds as a source of inoculum for biohydrogen production, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.07.006

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Please cite this article as: Cripa, F. B et al., Poultry slaughterhouse anaerobic ponds as a source of inoculum for biohydrogen production, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.07.006