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2014 32: 434Waste Manag ResRobles-González, N Ruiz-Ordáz, L Villa-Tanaca and N Rinderknecht-Seijas

KM Muñoz-Páez, HM Poggi-Varaldo, J García-Mena, MT Ponce-Noyola, AC Ramos-Valdivia, J Barrera-Cortés,Cheese whey as substrate of batch hydrogen production: Effect of temperature and addition of buffer

  

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Introduction

Cheese whey (CW) is a liquid that separates from the milk coag-ulation during cheese manufacture. CW contains most of the water-soluble components that are not integrated in the coagula-tion of casein. CW is considered a residue of the dairy industry and corresponds to around 85–90% of the total volume of pro-cessed milk, and its cost-effective utilization or disposal has become more and more important due to the legislative demands (Siso, 1996).

In Mexico, almost 53% of the milk production for pasteuri-zation and packaging is for fluid milk (SIAP, 2008) and the rest (47%) is used for dairy products such as cheese and yogurt. The generation of cheese represents 15% of national milk pro-duction; in 2007 the production of cheese in Mexico was 154,195 tonnes (Espinosa, 2009); it can be considered that the CW obtained varies from 4 to 11.3 kg fresh CW kg–1 cheese (Valencia, 2008). The CW produced in Mexico contains almost 50 thousand tonnes of potentially transformable lactose and 9000 tonnes of potentially recoverable protein (Carrillo Aguado, 2006).

CW is one of the polluting residues in the dairy industry that can negatively affect the environment and biological processes in wastewater treatment (Malaspina et al., 1996; Valencia-Denicia & Ramirez-Castillo, 2009), mainly because of: (a) its

high content of organic matter (biological oxygen demand, BOD: 30–50 g l−1 and chemical oxygen demand, COD: 70 g l−1); (b) the high content of lactic acid; and (c) low bicarbonate alka-linity (2.5 kg m−3 as CaCO3). Therefore there are a few reports of successful anaerobic (methanogenic) treatment (Ergüder et al., 2001; Mockaitis et al., 2006; Patel et al., 1999; Saddoud et al., 2007). Almost mandatory, the addition of alkalinity is required (Lo and Liao, 1986).

Cheese whey as substrate of batch hydrogen production: Effect of temperature and addition of buffer

KM Muñoz-Páez1, HM Poggi-Varaldo1, J García-Mena2, MT Ponce-Noyola3, AC Ramos-Valdivia3, J Barrera-Cortés3, IV Robles-González4, N Ruiz-Ordáz5, L Villa-Tanaca5 and N Rinderknecht-Seijas6

AbstractThe aim of this work was to evaluate the effect of buffer addition and process temperature (ambient and 35°C) on H2 production in batch fermentation of cheese whey (CW). When the H2 production reached a plateau, the headspace of the reactors were flushed with N2 and reactors were re-incubated. Afterwards, only the reactors with phosphate buffer showed a second cycle of H2 production and 48% more H2 was obtained. The absence of a second cycle in non-buffered reactors could be related to a lower final pH than in the buffered reactors; the low pH could drive the fermentation to solvents production. Indeed a high solvent production was observed in non-buffered bioreactors as given by low ρ ratios (defined as the ratio between sum of organic acid production and sum of solvents production). Regarding the process temperatures, no significant difference between the H2 production of reactors incubated at ambient temperature and at 35°C was described. After flushing the headspace of bioreactors with N2 at the end of the second cycle, the H2 production did not resume (in all reactors).

KeywordsCheese whey, hydrogen, batch fermentation, ambient temperature, phosphate buffer

1 Environmental Biotechnology and Renewable Energies R&D Group, Department Biotechnology and Bioengineering, CINVESTAV-IPN, México, México

2 Department Genetics and Molecular Biology, CINVESTAV-IPN, México, México

3 Department Biotechnology and Bioengineering, CINVESTAV-IPN, México, México

4 NOVA Universitas, Oax, México5ENCB-IPN, México D.F., México6ESIQIE-IPN, México D.F., México

Corresponding author:Héctor M Poggi-Varaldo, Department Biotechnology and Bioengineering, Environmental Biotechnology and Renewable Energies R&D Group, CINVESTAV-IPN, PO Box 14-740, Mexico DF 07000, México.Email: r4cepe@yahoo.com

527333WMR0010.1177/0734242X14527333Waste Management & Research XX(X)Muñoz-Páez et al.research-article2014

Original Article

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Muñoz-Páez et al. 435

Whey management has attracted more attention due to strin-gent legislation (Farizoglu et al., 2004) and economic considera-tions (Yang et al., 2007). From energy and wastewater treatment points of view, hydrogen fermentation of CW is an attractive alternative, although it is typically used as a first stage in a treat-ment process (Escamilla-Alvarado 2012; Muñoz-Páez et al., 2012a,b; Valdez-Vazquez et al., 2006).

There are different schemes of CW recovery: (a) use in fer-mentative processes, as culture medium for biomass, poly-(3- hydroxybutyrate), enzymes or biofuels production; (b) beverage manufacture; and (c) biofilm production (Cunningham, 2000; Fonseca et al., 2008; Valencia-Denicia and Ramirez-Castillo, 2009). Despite the technological developments for CW reuse and reclaiming, its disposal remains an important issue in the dairy industry.

The high content of organic matter of CW indicates that this waste can be an attractive substrate for biofuel production. In turn, hydrogen is one of the most attractive biofuels, because it is a clean energy carrier (Argun et al., 2008; Mizuno et al., 2002); it possesses an energy content per unit mass of 142 kJ g−1 or 61,000 Btu lb−1 (Das and Veziroglu, 2008), which is almost three times greater than that in some hydrocarbons (Ramachandran and Menon, 1998), and it is safer to use than domestic natural gas (Das and Veziroglu, 2008).

Lactose is the main contributor to the organic matter in CW. The maximum theoretical yield of H2 on lactose could be 8 mole per mole of lactose (Eq. 1), it is important to mention that this yield considers that 1 mole of glucose produced 4 moles of hydrogen (acetate pathway; Azbar et al., 2009; Valdez-Vazquez and Poggi-Varaldo, 2009a).

C H O H O H CO CH COOH12 22 11 2 2 2 3

5 8 4 4+ → + + (1)

There are factors that should be considered in order to obtain optimal H2 production from organic substrates, such as: (a) tem-perature; (b) pH; (c) substrate concentration; (d) type of the reac-tor; (e) alkalinity; (f) nutritional history of the cells; (g) method of inoculum enrichment; (h) method of methanogenesis inhibi-tion used; (i) hydraulic retention time; and (j) type of inocula, among others (Argun et al., 2008; Barros et al., 2010; Cavinato et al., 2011; Kothari et al., 2012; Valdez-Vazquez and Poggi-Varaldo, 2009b; Van Ginkel et al., 2001).

Regarding the operational and incubation temperature, it can affect: (a) the growth rate; (b) the metabolic activity of microor-ganisms; (c) the distribution of aqueous products; and (d) the substrate degradation (Lee et al., 2006; Mu et al., 2006). The optimal temperature for H2 production by mixed-culture systems can vary widely due to the presence of more complex bacterial populations (Yu et al., 2002). The selection of the temperature of operation should contemplate that the heat energy required to preserve higher operation temperatures can abate the net energy gain of biofuel production (Perera et al., 2012). Therefore it is interesting to study the H2 production at ambient temperature because it could save energy expenses such as on heating. It is

worth emphasizing that little is known about the dark fermenta-tion of CW at ambient temperature so far.

The pH can affect the microorganisms in several ways: (a) it impacts on the electrical charge of the cell membranes; (b) it could influence the uptake of nutrients; (c) it has an important effect on enzymatic activities, such as hydrogenasa; and (d) it determines product distribution in fermentation (Dabrock et al., 1992; Horiuchi et al., 2002; Li and Fang, 2007; Kataoka et al., 1997; Zhu and Yang, 2004). In H2 production, low pH can also inhibit hydrogen-consuming methanogenic microorganisms (Wu et al., 2010).

The decay of pH associated with low alkalinity and organic acid concentration imbalances might result in the arrest of H2 production (Lin and Lay, 2004). An alternative is to add suffi-cient buffer to compensate for the pH decrease resulting from the generation of organic acids (Zhu et al., 2009). Ferchichi et al. (2005) suggested that the H2 production could be increased by regulating the pH.

Thus, the aim of this work was to evaluate the effect of buffer addition and process temperature [two levels, ambient (A) and 35°C (M)], on H2 production in the batch fermentation of CW.

Materials and methodsExperimental design and bioreactors

The experimental design examined the effect of two factors on H2 production in batch fermentation (using CW as substrate), i.e. with phosphate buffer addition (PB) and the process temperature at two levels, ambient (15–22°C; average 18°C), where the bio-reactors were placed in a room without temperature control, and 35°C (in a walk-in chamber with temperature control). The experiments were run in duplicate. An abiotic control (sterilized inoculum and substrate) was also implemented. The main response variables were: cumulative H2 production (mmol H2gTS

−1), initial rate of hydrogen accumulation (mmol H2 (gTS h)−1), lag time for H2 production (h), the ratio A/B defined as the ratio between the acetic acid production and butyric acid produc-tion, and the ρ ratio (defined as the ratio between the sum of organic acid production and the sum of solvent production).

The bioreactors were glass vials of 60 ml capacity with 40 ml of working volume and 1 g of inocula (bioparticles of fluidized bed bioreactors) and 40 ml of 10 g CW l−1. After inoculation, the reactors’ headspace was flushed with N2 in order to maintain anoxic conditions. Then, the reactors were incubated at the cor-responding temperature. The fermentation was carried in batch mode with intermittent venting and headspace flushing according to the procedures reported by Valdez-Vazquez et al. (2006). During the fermentation, the biogas in the headspace was fre-quently released (intermittently vented) to maintain balance at an atmospheric pressure of 0.77 atm. When maximum H2 cumula-tive production was observed, the bottles’ headspace was vented and then one-time flushed with N2 to wash out the accumulated H2. Afterwards, the bottles were re-incubated; neither fresh inoc-ulum nor substrate was added.

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436 Waste Management & Research 32(5)

Inocula and substrate

The inocula were bioparticles from two anaerobic fluidized bio-reactors that produce H2 from CW. The bioparticles consisted of a medium support (particles of activated carbon, average diame-ter 1–2 mm) colonized by a microbial consortium (0.152 g VSS-biomass vial−1; Muñoz-Páez et al., 2012a). The structure of the reactors is similar to those of Garibay-Orijel et al. (2005) and Muñoz-Páez et al. (2012b). The organic carbon source was pow-der CW (PCW, 10 g l−1; 10.4 g COD l−1); the PCW characteristics are shown in Table 1. It could be considered sweet whey because the pH was in the range 5.9–6.6 (Bylund, 1995). The reactors without buffer (A and M) were fed with a synthetic wastewater with the following composition (in mg l−1; Leite et al., 2008; Zhang et al., 2007): CH4N2O (125); NiSO4.6H2O (1); FeSO4.7H2O (5); FeCl3.6H2O (0.5); CoCl2.2H2O (0.08); CaCl2.6H2O (47); SeO2 (0.07); KH2PO4 (85); K2HPO4 (21.7); Na2HPO4.2H2O (33.4); and NaHCO3 (1000). The buffer solution used in PB was composed of 24.5 g l−1 of KH2PO4 and 3.5 g l−1 K2HPO4.

Analyses

The main monitoring parameters were hydrogen concentration in the biogas, pH and soluble metabolites concentration. Hydrogen and methane contents in the biogas were analysed by gas chro-matography in a Gow-Mac chromatograph model 350 fitted with a thermal conductivity detector and molecular sieve 5A packed column. The temperature of injector, detector and column were set at 25°, 100° and 25°C, respectively. Argon was the carrier gas (Muñoz-Paéz et al., 2012b; Valdez-Vazquez et al., 2006). Cumulative hydrogen production (PH) was calculated from the values of biogas hydrogen concentration, and afterwards the effects of the experimental factors on PH were evaluated with an analysis of variance (ANOVA).

Soluble metabolites (volatile organic acids, lactic acid and solvents) were determined in the effluent, after filtration through a glass membrane filter. An aliquot of the filtrate was injected in a gas chromatography Varian Star 3400 equipped with a flame ionization detector for metabolite concentration determination. The injector and detector temperatures were set at 250°C. Nitrogen was used as a carrier gas with a 20 ml min−1 flow rate. The oven temperature was programmed as follows: 60°C for 2 min, increasing to 140°C at 5°C min−1, and then kept constant at

140°C for another 6 min. A 50-m-long, 0.32-mm internal diame-ter fused silica capillary column coated with 0.2 mm CP-Wax 57 CB was used.

Results and discussionEffect of buffer addition

The addition of buffer showed a positive effect on H2 production. A second cycle of H2 production (after the headspace flushing) was observed at both temperatures (Figure 1). The H2 production in the second cycle was approximately 60% of that in the first cycle alone (Table 2). The lag time was shorter than 24 h in all cases.

In the first cycle, the reactors with buffer (PB) had 1.3 times more cumulative H2 production than non-buffered ones (NPB; 1.85±0.14 and 1.37±0.07 mmol H2 gTS

−1, respectively; Table 2) and a lower initial rate of H2 accumulation (0.6 times) than NPB. A higher cumulative H2 production in PB compared with NPB could be due to a lower ∆pH (1.9; defined as pH initial−pH final) than ∆pH in NPB (2.8).

The high ∆pH (lower final pH) could affect the H2 producers, since the microorganisms could not adjust to the fast change (Khanal et al., 2004) and the fermentation could be driven to sol-vent production, to the detriment of H2 accumulation. No CH4 was observed in the biogas of our batch bioreactors. Neither H2 nor CH4 production was observed in the abiotic control.

Table 1. Main characteristics of powder cheese whey.

Characteristic Concentration

Fat (%) 0.85pH (%) 6.3Protein (%) 12.6Ash (%) 7.58Acetic acid (mg COD L−1) 54.8Propionic acid (mg COD L−1) 36.6Butyric acid (mg COD L−1) 40.8

COD: chemical oxygen demand.

Figure 1. Cumulative H2 production using cheese whey as sus-bstrate: (a) ambient temperature; (b) 35°C; ◊ without buffer; with buffer. Dotted lines indicate flushing of headspace with N2.

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Muñoz-Páez et al. 437

After flushing the headspace at the end of the second cycle with N2, the H2 production did not resume, i.e. there was no third cycle of fermentation for all bioreactors in this experiment.

Acetic acid (HAc), butyric acid (HBu), propionic acid (HPr), lactic acid (HLac), ethanol (EtOH) and butanol (BuOH) were the soluble microbial products (SMP) detected in this fermentation (Table 3). The ratio A/B has been used as an indicator of the met-abolic pathway favoured in the fermentative process. The thresh-old value of the ratio A/B (units of COD-equivalent) is 0.79: if the experimental A/B value is as high as 0.79 the H2 production via acetic acid is probably predominant, therefore if the experi-mental A/B value is less than 0.79 the H2 production via HBu is

probably predominant (Robledo-Narváez et al., 2013). In this work, the A/B ratios in all reactors were lower than the threshold value (0.79; Robledo-Narváez et al., 2013) and it can be said that the H2 production via HBu was probably predominant.

There was a significant difference in HBu production between buffered and non-buffered reactors. It is important to mention that the non-buffered HBu production was 1.3-fold higher than in the PB reactors despite there being only one cycle of hydrogen production in the non-buffered ones.

There are several studies that relate high concentration of HBu with low H2 production (Van Ginkel and Logan, 2005). The un-dissociated HBu was related to the initiation of

Table 2. Hydrogen production, initial rate of hydrogen accumulation, pH and ∆pH of the fermentation of CW.

Reactor PH (mmol H2 gTS−1) Ri,H

(mmol H2(gTS h)−1)Initial pH

Final pH ∆pH

1 cycle 2 cycle Total p-value 1 cycle p-value 2 cycle

A-NPB 1.37±0.14 – 1.37±0.14 NA 0.031 NA – 7.39 4.70±0.08 2.70±0.08A-PB 1.71±0.02 1.35±0.15 3.07±0.13 NA 0.025 NA 0.028 5.9 4.02±0.03 1.88±0.03M- NPB 1.36±0.01 0.15±0.13 1.51±0.12 NA 0.040 NA – 7.39 4.47±0.01 2.93±0.01M-PB 1.99±0.03 0.96±0.46 2.95±0.13 NA 0.016 NA 0.02 5.9 3.99±0.02 1.92±0.02PBa 1.85±0.20 1.16±0.28 3.01±0.08

0.00080.035

<0.00010.024 5.9 4.00±0.03 1.90±0.03

NPBb 1.37±0.001 0.07±0.10 1.44±0.10 0.021 – 7.39 4.58±0.14 2.81±0.14Ac 1.54±0.25 0.68±– 2.22±1.20

0.92780.023

<0.00010.014 6.65 4.36±0.39 2.29±0.47

Md 1.68±0.45 0.55±– 2.23±1.02 0.032 0.01 6.65 4.23±0.28 2.42±0.58

A-NPB: ambient temperature without buffer; M-NPB: 35°C without buffer; A-PB: ambient temperature with buffer; M-PB: 35°C with buffer. PH: cumulative H2 production; Ri,H initial rate of hydrogen accumulation. aAverage result calculated by pooling A-PB and M-PB results. bAver-age result calculated by pooling A-NPB and M-NPB results. cAverage result calculated by pooling A-NPB and A-PB results. dAverage result calculated by pooling M-NPB and M-PB results. The lag time Tlag for hydrogen production onset in all reactors was <24 h. p-value: It is the probability of getting an F-value of this size if the term did not have an effect on the response.

Table 3. Acid and solvent concentration of hydrogen production with cheese whey.

Reactor concentration (mg COD l−1)

A-NPB A-PB M-NPB M-PB

Acetic acid 1 308±207 1 675±591 1 966±232 1 462±883Propionic acid 523±105 1 784±845 400±60 671±43Butyric acid 4 362±866 3 136±125 4 378±255 3 464±304Lactic acid ND 243.4±43.5 ND 229.7±4.28Acetone ND ND ND NDMethanol ND ND ND NDEthanol 303±161 55.1±14.7 128.2±26.1 173.0±26.0Butanol 1 422±186 56.5±41.8 216.5±47.1 86.7±38.1EtOH/SMP (%) 2.4±2.1 0.8±0.2 1.8±0.3 2.9±0.7BuOH/SMP (%) 9.6±9.6 1.2±1.1 3.0±0.5 1.5±0.8HAc/SMP (%) 22.3±9.2 28.8±1.6 26.9±2.9 30.2±1.2HPr/SMP (%) 16.8±15.8 27.5±15 5.1±0.7 10.5±1.7HBu/SMP (%) 51.7±4.0 48.8±5.0 61. 2±3.7 56.5±8.3A/B 0.28±0.04 0.54±0.18 0.45±0.1 0.6±0.3TVOA 6 193±345 5 702±1 281 6 743±259 6 102±600SMP 7 919±214 5 776±1 270 7 088±326 7 241±427ρ 3.6±0.5 79.2±27.1 19.9±2.9 23.6±7.3

A-NPB: ambient temperature with no buffer addition; A-PB: ambient temperature and buffer addition; M-NPB: mesophilic (37oC) with no buf-fer addition; M-PB: mesophilic incubation and buffer addition; A/B: acetic to butyric acid ratio. EtOH: ethanol; BuOH: butanol; HAc: acetate; HPr: propionate; HBu: butyrate; TVOA: total volatile organic acids=HAc+HPr+HBu; SMP: soluble microbial products=TVOA+EtOH+BuOH. Based in COD l−1. Conversion factors f (mgCOD mg product−1): EtOH=1.50; BuOH=2.59; HAc=1.07; HPr=1.51; HBu=1.81; HLac=1.6. COD: chemical oxygen demand.

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438 Waste Management & Research 32(5)

solventogenesis (Husemann and Papoutsakis, 1987) and in our work the ρ ratios in non-buffered reactors were less than in PB, indicating a high solvent production. It is known that butyrate production regenerates NAD+ through both H2 production and butyryl phosphate reduction, but, if the butyrate production pathway is inhibited by excess butyrate and if H2 production is disadvantageous because of high concentrations of dissolved hydrogen, the only electron sink left is by solvent production, which does not result in any H2 production (Van Ginkel and Logan, 2005). Van den Heuvel et al. (1992) determined that the critical inhibitory concentration of un-dissociated HBu was 50 mM for a metabolic switch to solventogenesis; in our work, the unbuffered reactors exhibited a HBu concentration nearly that value (41.02±0.5 mM) in the first cycle, whereas the PB reac-tors produced 31.2±2.6 mM after the second cycle.

Effect of temperature

Regarding the process temperature, there was no significant dif-ference between the cumulative H2 production, concentration of organic acid and ρ of reactors incubated at ambient temperature and at 35°C.

Several authors reported the highest H2 production at higher temperatures (Akutsu et al., 2009; Lee et al., 2006; Lin et al., 2008; Youn and Shin, 2005). Yet, in our work, we obtained a good performance at ambient temperature. It is important to men-tion that the inocula of batch fermentation come from fluidized bed reactors that had been operated with sucrose; during the operation with sucrose the continuous bioreactors at ambient temperature also showed a better performance than at 35°C (Muñoz-Páez et al., 2012b).

The favourable H2 production obtained at low temperature is in agreement with that obtained by Infantes et al. (2011), who investigated the influence of pH and temperature on acidogenic fermentation and bio-hydrogen production in batch fermentation, using glucose as substrate. They suggested that the net effect of the low pH and high temperature causes a higher inhibition due to the high concentration of un-dissociated organic acids. The increase in temperature also induced two opposite effects: (a) it elevated the microbial metabolism and (b) increased membrane permeability (Bischof et al., 1995; Zoetemeyer et al., 1982).

Table 4 shows the H2 production of several systems that used CW as substrate and it can be observed that the yield of our work (3.81 mmol gTS

−1 at ambient temperature and 2.95 mmol gTS−1 at

35°C) is in the middle of the reported range.Referring to the results achieved at ambient temperature, the

highest H2 production corresponded to A-PB (3 mmol H2 gTS−1),

this value was near to the highest yield of 3.5 mmol H2 gTS−1

observed by Azbar and Dokgoz (2010) (Table 4). They produced H2 in a continuous fermentation at 55°C and a pH of 5.5; the substrate was raw CW (RCW) diluted with distilled water and subjected to heat shock treatment (105°C/5 min). They added supplements such as (g l−1): NaHCO3 (1.25), NH4Cl (2.5), KH2PO4 (0.25), CaCl2 (0.25), NiSO4 (0.032), MgSO4.7H2O (0.32), FeCl2 (0.02), Na2MoO4.2H2O (0.0144), ZnCl2 (0.023), CoCl2.6H2O (0.021), CuCl2.6H2O (0.01), MnCl2.4H2O (0.03), yeast extract (0.05) and cysteine (0.5). Our results are encourag-ing because we obtained almost the same results but without heat shock treatment to the CW and supplement addition.

In this research, the lowest value of H2 production (1.37 mmole H2 gTS

−1) was achieved at ambient temperature without phosphate buffer (A-NBP). The value is in the middle range of

Table 4. Hydrogen production using cheese whey as substrate.

Inocula Conditions Substrate volumetric loading rate (g COD (l day)−1)

Y’ hydrogen pseudoyield (mmol H2 gTS

−1)

Soluble microbial products

Ref.

Sludge heat shock treatment (85°C/45 min)

HRT:a 1, 2 and 3.5 days, T: 55°C, pH 5.5, continuous

Fresh raw cheese whey with heat shock treatment (105°C/5 min), 47

0.91 Acetate, butyrate, propionate, isobutyrate, isocaprionate, formate, lactate

1

Acidogenic lab-scale reactor

HRT=12 h, pH=5, T= 30°C, continuous

Cheese whey stored at 4°C, 20

0.18 Acetate, butyrate, lactate and valerate

2

Digestates of a CSTRb HRT=24 h, T: 35°C, pH: 5.2, continuous

Cheese whey stored at -20°C, 30

1.5 Acetate, butyrate, lactate

3

Anaerobic mixed microflora from an UASB,c heat shock treatment

HRT=24 h, T: 55°C, pH=5.5, continuous

Cheese whey with heat shock pretreatment, 85°C/30 min, 30

3.5 Lactate, butyrate 4

Digestates of anaerobic reactor with heat shock treatment (90°C, 1 h)

HRT=24 h, T: 25°C, pH=4.1, continuous

Powder, 10.04 1.4 Acetate, propionate, butyrate, ethanol, butanol, lactate

5

Bioparticles of AFBRd Batch, T: 25°C; 35°C, pH: 5.9

Powder, 10.04,e with phosphate buffer

3.07, at 18 °C, 2.95, at 35°C

Acetate, propionate, butyrate, ethanol, butanol, lactate

6

a Hydraulic retention time; bcontinuous stirred tank reactor; cupflow anaerobic sludge blanket reactor; danaerobic fluidized bed reactor; TS: total solids.COD: chemical oxygen demand. References: 1. Azbar et al. (2009); 2. Castelló et al. (2009); 3. Venetsaneas et al. (2009); 4. Azbar and Dokgoz (2010); 5. Muñoz-Páez et al. (2012b); 6: this study. eSubstrate concentration (g COD l−1).

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Muñoz-Páez et al. 439

other works using CW as a substrate for H2 production (0.18–3.5 mmole H2 gTS

−1, Table 4). Our result is lower than that obtained by Azbar and Dokgoz (2010), possibly due to the high ∆pH (2.69 units) since we did not add buffer. In contrast, in the test of Azbar and Dokgoz (2010) pH control was implemented so their ∆pH was lower.

Conclusion

•• The reactors with phosphate buffer showed a second cycle of H2 production after the headspace flushing whereas bioreac-tors without buffer only exhibited a first cycle of H2 generation.

•• Hydrogen production in the second cycle of PB reactors was approximately 60% that in the first cycle alone. The initial rate of H2 accumulation in the second cycle was lower than in the first one at both temperatures.

•• Regarding the process temperature, there was no significant difference between the H2 production of units incubated at ambient temperature and at 35°C.

The use of CW as substrate for biofuels (H2) could impact waste-water management in the dairy industry, because only 47% of the overall CW production worldwide and just 10% of the CW in Mexico is reused and reclaimed. On the other hand, operation at ambient temperature could help to decrease the energy expendi-ture in the hydrogenogenic fermentation.

AcknowledgementsThe authors wish to thank SECITI-GDF for support with projects PICCO 10-27 and PICCO 10-28 and CINVESTAV del IPN for finan-cial support to this research, and CONACYT for a graduate scholar-ship to KMM-P. The excellent help of Professor Elvira Ríos Leas, Mr Cirino Chávez-Rojas and Mr Gustavo Medina (Central Analítica) as well as Mr Rafael Hernández-Vera from the GBAER-EBRE Group, CINVESTAV del IPN, is gratefully acknowledged.

Declaration of conflicting interestsThe authors declare that there is no conflict of interest.

FundingThis work was supported by the projects ICYTDF now SECITI-GDF [PICCO 10-27 and 10-28] and CINVESTAV del IPN, CONACYT granted a graduate scholarship to KMM-P.

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