1 Biohydrogen production by chemical-decoupling of the anaerobic

2 fermentation of agave vinasses

4 A. Bocanegra-Martínez 1, G. Jiménez-García 2, J.A. Cortés 1, E.D. Alanis-Silva 1,

5 R. Maya-Yescas *,1

1
7 Facultad de Ingeniería Química, Universidad Michoacana de San Nicolás de Hidalgo

8 Ciudad Universitaria, 58030, Morelia, Michoacán, México

2
9 Departamento de Ingeniería Biomédica, Instituto Tecnológico Superior de Pátzcuaro

10 Av. Tecnológico # 1, Tzurumútaro, 61615, Pátzcuaro, Michoacán, México

11 (*) corresponding author: rmayay@umich.mx

12

13 Abstract

14 This work deals with the production of biohydrogen by anaerobic fermentation of agave

15 vinasses, using as inoculum pre-treated sludge from a UASB wastewater treatment plant and

16 supplementing nitrogen and phosphorus by wastes from the seafood food industry. The effects

17 of the initial organic loading (COD) of the substrate and the initial pH on biohydrogen

18 production were determined following the anaerobic digestion of vinasses in 125 ml Erlen-

19 Meyer Flasks by a factorial 22 design. The highest production of biohydrogen was obtained

20 using initial pH of 3.5, with initial organic substrate load of 8,000 mg/L. Subsequently, using

21 the above operating conditions, the fermentation was scaled up to 3L UASB bioreactor and a

22 maximum yield in biohydrogen production of 1.1643 (mol H2)/(L of substrate) was reached.

23 This yield is comparable with data reported in the literature when vinasses from another type

24 of source has been used as a substrate. The results obtained evaluate the potential of mezcal

25 agave vinasses for biohydrogen production.

26
27 Keywords: Anaerobic fermentation, agave vinasses, decoupling metabolic pathways,

28 biohydrogen production.

29

30 1. Introduction

31 The search for renewable sources for the production of fuels is undoubtedly one of the most

32 current research activities worldwide today. This is driven by multiple factors such as global

33 warming of the planet, diplomatic problems due to limited reserves of hydrocarbons and the

34 unequal distribution of non-renewable resources among different countries. With these new

35 renewable energy sources, it is intended to stop the excessive use of fossil fuels (natural gas,

36 coal and petroleum) and consequently reduce the environmental impact generated by their

37 combustion.

38

39 Among the possible renewable fuels is biohydrogen, a carrier of clean energy, since its

40 oxidation reaction with oxygen produces 120 kJ/g of energy, releasing only water as waste.

41 Although this element is not freely found on the biosphere, it is one of the most abundant

42 elements of the earth, since it forms numerous chemical compounds such as water and biomass.

43

44 During last decade, about 95% of biohydrogen production in the world was obtained from fossil

45 fuels, through synthesis gas production, as in the case of steam reforming of natural gas or light

46 naphtha, coal gasification and oxidation of heavy oil fractions. The other 5% biohydrogen was

47 obtained by electrolysis of water (4%) and biomass gasification (1%) (Balat Kirtay, 2010).

48 However, in order to suspend the use of non-renewable resources and to lower the current costs

49 of biohydrogen production, new ways of obtaining it have been developed through

50 thermochemical processes, photobiological processes, biomass gasification and new water

51 electrolysis technologies, among others (Kalamaras and Efstathiou, 2013).

52

53 One of the alternatives that continues generating high expectations is biohydrogen production

54 (renewable energy source) from biomass conversion routes through biological processes such

55 as anaerobic fermentation. This process consists of the digestion of organic material by means

56 of microorganisms in the absence of oxygen (Figure 1). Among the advantages of this

57 technology are the ability to use any carbohydrate-rich substrate, biohydrogen production rates

58 faster than those of other biological processes, lower operating costs than other methods that

59 require light energy, and the microbial communities used are available in waste waters,

60 anaerobic compost or deep sludge (Manish and Banerjee, 2008). This has been possible thanks

61 to the implementation of systems that separate the hydraulic retention time (HRT) from the cell

62 retention time (CRT), which have been called “high-rate bioreactors”. During this process,

63 combustible biogas, and sludge with properties suitable for use as fertilizers, are obtained.

64

65

66 Figure 1. Stages of the anaerobic digestion process.

67

68 Several factors influence the methodology of the treatment of waste waters. Some of them

69 include environmental conditions, operating conditions, mesophilic temperature regime (25,

70 40)° C may be more attractive than thermophilic temperature regimes, mainly due to lower

71 energy requirements during the process, and bioreactor configuration. In this respect, the flow

72 of activated sludge in a bioreactor, normally considered as the influence of flocs, constitutes

73 one of the most critical phenomena in the treatment and physiology of a microbial community.

74 Anaerobic digestion is affected by specific characteristics of feed substrate, operational

75 parameters such as hydraulic retention time (HRT) and organic load rate. Environmental

76 factors, such as presence of inhibiting or toxic substances, temperature and incommensurable

77 variables play secondary but significant roles.

78

79 During the last years, the popularity of mezcal consumption has grown in Mexico; mezcal is

80 produced only in few states in the country that have the “designation of origin”, such as

81 Michoacán, Oaxaca, Guerrero and Guanajuato. The production of mezcal generates 74% of

82 waste vinasses in its production process. It is estimated that in previous years, generated 14 to

83 27 million L of mezcal vinasses; approximately 80% of the volume is discharged as vinasses

84 directly into bodies of water (rivers, lakes and reserves), to the municipal sewer system, or to

85 the ground without receiving the appropriate treatment for their disposal. The last practice

86 causes soil deterioration as consequence of its high salt content, the presence of substances such

87 as acetic acid, lactic acid, glycerine and ammoniacal nitrogen can poison crops (phytotoxicity).

88 The use of this waste as an alternative source of higher value-added materials would be

89 advantageous from an economic and environmental point of view (Moya, 2001).

90

91 Motivated by the successful biohydrogen production from anaerobic fermentation using tequila

92 and nejayote vinasses (García-Depraect et al., 2017), the goal of this work is to use agave

93 vinasses to obtain biohydrogen. Tequila is produced from only one species of agave, the “Blue

94 Variety Agave” Tequilana weber, which is widely studied. On the other hand, for the
 95 production of mezcal more than a dozen of different types of agaves are used. As consequence,

96 agave industrial processing is not as standardized as tequila processing, introducing challenges

97 because of feed properties variability in vinasses treatment processes. For example, mezcal

98 vinasses contain variable proportions of sugar oligomers and some waxes, absent in tequila

99 ones, which influence treatment conditions because of different microbial growth rates.

100

101 Alone, nutrients in agave vinasses are insufficient to support the microorganism consortia

102 present in anaerobic fermentation, then another waste coming from the sea food industry was

103 used to compliment the feedstock to the anaerobic fermentation process. Mainly it consists of

104 shrimp and fish waste and also contains vegetable residues with which the food was prepared.

105 This blend showed by elemental analysis, to be rich in minerals.

106

107 Pohland and Ghosh (1971) were the first to propose the physical separation of acid-forming and

108 methane-forming microbial populations, in two separate bioreactors in series, where the optimal

109 conditions are created for each group of microorganisms, thus improving the stability and

110 control of the process. The first bioreactor would be used for the production of volatile fatty

111 acids (VFA) and the second bioreactor for methane production. This configuration overcomes

112 the limitations of the conventional method of anaerobic digestion, increasing the robustness of

113 the system, facilitating better application, control and optimisation of the process (Dareioti et

114 al, 2009). It also allows the selection and enrichment of the different groups of microorganisms

115 in each separate bioreactor, increasing the stability of the process by better control of the

116 acidogenic phase, preventing overload and inhibition by toxic compounds. On the other hand,

117 the first bioreactor acts as pH buffer to the methanogenic populations in the second bioreactor.

118
119 Many published works show considerable discrepancies in inhibition and toxicity levels at the

120 methanogenic stage for different substrates. The main reason for this phenomenon is due to the

121 complexity of microbial interactions during the process of anaerobic digestion, where some

122 mechanisms, such as antagonism, synergism, acclimatization processes, determine inhibition

123 phenomena (W.M. Chen, et al. 2005). A large number of substances inhibit the process of

124 anaerobic digestion, which prevents adequate bacterial growth, causing a deterioration in the

125 stability of the system and the accumulation of acidic species; however, limited research has

126 been carried out on the inhibition of the acid phase. However, it is possible for the micro-

127 organisms present in the anaerobic bioreactors to adapt to these compounds during the

128 anaerobic digestion process, provided that they do not exceed the permissible values for their

129 positive effect on biochemical processes; therefore, the concentrations at which they are found

130 and the appropriate population balance must be taken into account, the latter being ensured in

131 the granulation of the anaerobic sludge from the advanced UASB reactors.

132

133 This project evaluates the possibility of increasing production of biohydrogen by anaerobic

134 digestion of agave vinasses by decoupling fermentation stages, in a scheme of two bioreactors

135 in series:

136

137  The first bioreactor is attempted to for the hydrolysis-acidification stages, which is

138 followed by the decrease in pH and biohydrogen production.

139  The second bioreactor is regulated for the acetogenesis-methanogenesis phase, by the

140 addition of chemical acetate nutrients, which is followed by the partial stability of pH and

141 the production of some biogas.

142
143 This treatment scheme provides some advantages compared to that performed in the same

144 bioreactor, including greater process stability, greater resistance to inhibitory compounds and

145 controlled acidification plugging.

146

147 2. Materials and methods

148

149 2.1. Vinasses from agave mezcal and characterization

150 It is during the last stage of mezcal production that the vinasses are generated, which are the

151 residual product of the distillation of the fermented must once the alcohol-rich components are

152 separated. Part of their volume and variability of organic load comes from other minor effluents

153 such as the cleaning water of fermentation vats, which contributes significantly to the load

154 having values of around 25,000 mg of Chemical Oxygen Demand per litre (COD/L), and

155 cooling water of the condensers, which dilutes and increases the volume of vinasses.

156

157 The physicochemical characterization of the agave vinasses (Table 1) showed that agave

158 vinasses are rich in carbohydrates, 11.3%p of reducing sugars and 1.14%p of glucose. For this

159 reason, the agro-industrial waste of mezcal agave vinasses stands out as a material that could

160 be fermented for biohydrogen production or other metabolites of interest.

161

162 Table 1. Physicochemical composition of the agave vinasses used substrate.

Property Average amount Standard deviation Unit

Humidity 86.14 1.43 %

Ashes (wet base) 1.31 0.24 %

Reducing sugars (1g vinasses) 0.113 0.002 g/L

Glucose (40g vinasses) 0.455 0.029 g/L

 pH 3.50 0 pH

Nitrogen (wet base) 0.58 0 %

Phosphorus (wet base) 0.05 0 %

Iron (wet base) 23.5 10.6 mg/kg

163

164 2.2. Characterization of nutrients supplement and inoculum sludge

165 In order to fulfil nutrient requirements and to improve the yield to biohydrogen production (Pan

166 et al., 2008; Lin and Lay, 2005), a supplement, based on waste from the seafood industry, was

167 added to the agave vinasses substrate (Table 2).

168

169 Anaerobic sludges extracted from a UASB reactor of an anaerobic wastewater treatment plant

170 (GEA Ambiental S.A. de C.V., located in Morelia, Mexico), were heat treated at 303.15 K

171 before to be used as inoculum. Likewise, a physical characterization (Table 3) was carried out

172 according to Standard Methods (Woo and Song, 2010; APHA, 1999).

173

174 Table 2. Mineral description of the nutrients supplement.

Amount Amount
Nutrient Component
(mg/L) (mg/L)

Urea 4,000 Na2MoO4 10

Peptone 3,000 CoCl2·6H2O 200

Na2HPO4·12H2O 21,500 AlK(SO4)2·12H2O 10

KH2PO4·2H2O 8,100 NiCl·6H2O 1

MgCl2·6H2O 100 N[(CH2)COOH]3 2,000

FeSO4·7H2O 200 Vitamin B12 10

 L-cysteine 100 Vitamin C 25

MnSO4·7H2O 10 Citric acid 20

ZnSO4·7H2O 50 Folic acid 10

H3BO3 10 Creatine 25

CaCl2·2H2O 10 p-aminobenzoic acid 10

175

176 Table 3. Physical characteristics of the inoculum.

Total sedimentable Volatile Suspended Total Suspended Apparent

solids Solids Solids density

(mL sludge/mL sample) (g/L) (g/L) (g/mL)

7.5/35 1.7086 2.3257 0.0109

177

178 2.3. Design of experiments at flask level

179 An experimental factorial 22 design was carried out in 125 mL Erlen Meyer flasks, with a

180 working volume of 100 mL, running two replicas in each experimental point. The first factor

181 considered was the initial concentration (low level= 5 g glucose/L, high level= g glucose/L)

182 and the second one addition or no addition of co-substrate respectively. The production of

183 biohydrogen was the response variable.

184

185 Previous upon inoculation, samples were prepared with the stillage and nutrient co-substrate

186 mixture, the appropriate amount of water was added to adjust to the appropriate COD (5,000

187 mg / L), the substrate and the nutrient mixture They were heated to 308.15 K to obtain an

188 anaerobic atmosphere in each flask. Then, the samples were inoculated in such a way to obtain

189 a 20% inoculum concentration (v/v), the pH was adjusted to the desired value of 3.5, and the

190 temperature of the incubator was adjusted to 308.15 K, without stirring.

191

192 2.4. Anaerobic fermentation in the decoupled UASB reactor

193 Fermentations were carried out in two UASB type reactors of 3 L capacity, connected in series,

194 using effective working volume of 2 L. The temperature was maintained at 306.15 K by means

195 of a thermal bath (Thermo Scientific ™). Initial values of pH of 3.5, COD of 5,000 mg/L and

196 the nutrients supplement amount necessary to fulfil initial minerals requirement were used

197 (Table 4). Hourly sampling was carried out, through a hole located in the bioreactor cover, for

198 48 hours.

199

200 Table 4. Characterization of the substrate used in anaerobic fermentation.

Variable Value

Initial pH of the mixture 4.27

DQO (mg/L) 13,100

Total Nitrogen (mg/L) 1,300

Total phosphate (mg/L) 7,600

201

202 2.5. Analytical methods

203 The quantitative analysis of biohydrogen in gas can be determined based on Liebig’s technique

204 for elemental analysis for organic compounds. This method was invented by Joseph Louis Gay-

205 Lussac ; Justus von Liebig studied the method while working with Gay-Lussac between 1822

206 and 1824 and improved the method in the following years to a level that it could be used as

207 standard procedure for organic analysis (He X. et al., 2005). The physicochemical

208 characterization of agave vinasses was based on standard methods (Table 5).

209

210
211 Table 5. Methods used for the physicochemical characterization of mezcal agave vinasses.

Assay Method / Norm

DQO (mg/L) Method 8000 DE Hach Digestion- spectrophotometry

Total Nitrogen (mg/L) Method 8075 DE Hach Digestion- spectrophotometry

Total Phosphate (mg/L) Method 10127 DE Hach Digestion- spectrophotometry

Suspended Solids (mg/L) NMX-AA-034-SCFI-2001 gravimetry

Total Solids (mg/L) NMX-AA-004-SCFI-2000 gravimetry

pH NMX-AA-008-SCFI-2000 pH-metro

212

213 2.6. Kinetic model of the biohydrogen production

214 The kinetic model used to describe the experimental data is based on the ADM-1 anaerobic

215 digestion model (X. Zhao, 2011) and the modified Gompertz equation (2).

216

𝑅𝑚 ∗ 𝑒
𝐻 = 𝑃𝑚𝑔 𝑒𝑥𝑝 [ (𝜆 − 𝑡) + 1] (2)
𝑃𝑚

217

218 Here, H is the cumulative amount of H2 produced (mol), after t (h) incubation hours, Pm is the

219 amount of the maximum potential of H2 produced (mol), e is the Napier’s constant (2.71828),

220 λ is the time required to start the production of H2 (h) and Rm is the maximum production rate

221 of H2 (mol/h). The kinetic constants (Pm, Rm and λ) were obtained by adjusting the model to

222 the experimental data by nonlinear regression, using Polymath ™ software version 6.1.

223

224 3. Results and discussion

225

226 3.1. Analysis of the biogas mixture

227 After stabilization by 60 days no significant amounts of methane were detected in the analysis
228 of the gases produced during the fermentation process (Figure 2). This fact indicates that the

229 thermal pre-treatment carried out in the system, in addition to the low pH used in the tests, are

230 effective in favouring selective biohydrogen production.

231

70

60

50
Concentration (%)

40
Methane

30 Hydrogen
Other *

20

10

0
20 45 60
Time (Days)
232

233 Figure 2. Composition of the gas produced by the UASB reactor.

234 * Other: H2S, CO2 and O2.

235

236 3.2. Kinetic parameters of biohydrogen production

237 After collecting kinetic data, the kinetic parameters (Rm, λ and Pm) were calculated according

238 to the modified Gompertz equation (2), and the average and the standard deviation for each

239 condition were analysed (Table 6). Experimental runs 5, 7 and 8 were the ones that reach the

240 highest biohydrogen production (Pm). However, there is a notable difference in the start time

241 of biohydrogen production (λ) between runs 5 and 7 (7.398 h and 7.906 h, respectively), and 8

242 (λ = 22.974 h). It is also noticed that in runs (5, 6, 7 and 8) that were carried out with the addition

243 of the nutrient formulation, more biohydrogen was produced than in the case of the runs without
244 addition of nutrients (Table 7).

245

246 Table 6. Estimated kinetic parameters for the fermentations performed.

Parameter / Conditions according to the standard design matrix

Run 1 2 3 4 5 6 7 8

Average

Pm (mmol) 0.648 0.669 0.844 0.605 1.188 0.824 1.145 1.201

Rm (mmol/h) 0.093 0.119 0.143 0.096 0.161 0.032 0.145 0.052

λ (h) 6.972 5.601 5.889 6.317 7.398 26.123 7.906 22.970

Standard deviation

Pm (mmol) 0.028 0.058 0.038 0.052 0.102 0.058 0.019 0.032

Rm (mmol/h) 0.038 1.279 0.002 0.114 0.000 0.004 0.020 0.003

λ (h) 0.182 0.869 0.615 0.662 0.616 1.416 2.751 0.087

247

248 Table 7. Yield to biohydrogen, effects and interactions.

YH2
Run <YH2> Interaction or Effect
Replica I Replica II

1 0.0251 0.0250 0.0250 µ = 0.03180

2 0.0261 0.0298 0.0280 E1 = 0.00364

3 0.0229 0.0184 0.0206 E2 = -0.00861

4 0.0122 0.0328 0.0225 E3 = 0.01548

5 0.0513 0.0455 0.0484 I12 = 0.00494

6 0.0443 0.0415 0.0429 I13 = 0.00124

7 0.0262 0.0253 0.0257 I23 = -0.00369

8 0.0418 0.0403 0.0410 I123 = 0.00549

249

250 To perform the statistical analysis of the experimental design, the yield to biohydrogen

251 production (YH2), which units are (mol H2 produced)/(mol of glucose consumed), was used as

252 response variable. This was calculated by dividing the kinetic constant of maximum

253 biohydrogen production (Pm) among the moles consumed of glucose in each run. Table 7 shows

254 the calculated values, as well as the effects and interactions obtained in the statistical analysis.

255 With the data of the variances between experiments a confidence-interval of statistical non-

256 significance was calculated at 95% confidence value, which turned out to be [-0.0064; 0.0064].

257 Under these results, the effects of variables 2 (initial substrate concentration) and 3 (food waste

258 supplement) are significant. Effect 1, associated with the initial pH of the mixture, was not

259 significant in this study.

260

261 In the case of effect 2 (-0.00861) the negative sign indicates that, at lower initial substrate

262 concentrations, the yield to biohydrogen production is favoured. This phenomenon has been

263 widely reported in the literature and is explained due to inhibition processes both by the

264 substrate used, and by products generated during the fermentation process (acids, alcohols and

265 even biohydrogen itself).

266

267 The value of effect 3 (0.01548) indicates that if the nutrient formulation is used, higher yields

268 to biohydrogen production are obtained. In addition, this effect exhibits the highest absolute

269 value, so it becomes the most influential effect on biohydrogen production. The nutrient

270 formulation is composed of metallic elements such as Zn, Na, Fe and Mn, which help in

271 transport and on cofactors of enzymes, improving biohydrogen production in comparison to

272 those carried out without addition of nutrients to fermentation media.

273
274 3.3. Biohydrogen production in the decoupled UASB reactor

275 The process was running out under the conditions corresponding to the run 5 of the previous

276 stage. This is due to the fact that under these conditions the highest yield was obtained (0.0484

277 mol H2/mol glucose consumed) and because it meets the conditions deduced as favourable from

278 the statistical analysis: variable 2 at its lower level (5 g glucose/L) and variable 3 at its highest

279 level (including nutrients supplement). The initial pH was set to the lower level of 3.5.

280

281 The data of biohydrogen production (Figure 3) show the lag time in biohydrogen production in

282 the bioreactor to be approximately 48 h; this time is after the stabilization of the system (20

283 days). The maximum biohydrogen production was 36.7 mol, which corresponds to a yield to

284 hydrogen of 1.54 (molH2/mol of substate consumed). This yield is higher than the one obtained

285 in the run 5 of the previous stage, which could be consequence of the better mass transfer in the

286 3 L bioreactor, due to the mechanical agitation. The increasing of hydrogen production was

287 because microorganisms were able to take advantage of 8% more of the initial substrate.

288

500
400
vOL. H2 (ML)

300
200
100
0
0 16 32 56 64 74 82 90 98 104 108 116 120
Time (D)
289

290 Figure 3. Accumulated biohydrogen obtained in the UASB reactor.

291

292 Kinetic parameters of the kinetic model of hydrogen production were adjusted for the results in

293 the UASB reactor. It should be noted (Table 8) that these parameters are acceptably close to
294 those obtained in run 5, whereas the stabilization of the system took longer, affecting the λ

295 variable in a sensitive way. The global effect of the stabilization time seems to predict a slower

296 process, however once production of hydrogen begins, agitation enhancing of mass transfer

297 favours higher and faster use of the substrate.

298

299 Table 8. Kinetic parameters obtained for the UASB reactor.

Pm (mmol) Rm (mmol/h)  (h)

Experimental value 1.776 0.111 16.0

Relative difference -1.01% -31.06% +116.22%

300

301 For anaerobic fermentations, where acidogenic bacteria such as Clostridium sp. are used

302 primarily, there is a relatively low theoretical yield to biohydrogen, 4 (mol H2)/(mol of glucose).

303 The yield obtained in this study was 1.54 (mol H2)/(mol of glucose consumed) and represents

304 38.5% of the theoretical value that could be achieved, falling within the range of yields that

305 have been obtained in other studies reported in the literature (Table 9).

306

307 Table 9. Comparison of some H2 yields obtained in anaerobic fermentations in systems.

Yield

Substrate Microorganism (mol H2/mol of Reference

substrate consumed)

Saccharose Clostridium butyricum 2.78 Cheng et al., 2005

Hexose added Anaerobic sludge 1.29 Jung et al., 2010

Glucose Clostridium beijerinckii 1.97 Zhao et al., 2011

Glucose Thermotoga 0.53 Pan et al., 2008

Glucose Clostridium butyrium 2.02 Jiang et al., 2016

 Agave vinasses Anaerobic sludge 1.54 This study.

308

309 Chemical oxygen demand (COD) analysis was performed to the fermentation residue, in order

310 to determine the characteristics of the eventual waste streams of this process. The value

311 obtained was 8,000 mg/L, evidencing high content of organic matter still available. These

312 mixtures, due to their rich content of carboxylic acids, could be used to produce more

313 biohydrogen through photo fermentative processes.

314

315 4. Conclusions

316  This research showed that separation of the stages in this type of bioreactor has a positive

317 influence on biogas production, with hydrogen being the most favoured during the first

318 stage. However, the study of decoupling and operating UASB type reactors is still in early

319 stages.

320  The sludge heat treatment and the pH used were effective in preventing the growth of

321 methanogenic vegetative bacteria, because no significant methane was detected as a

322 secondary product.

323  The mezcal agave vinasses residue stands out as a carbohydrate-rich material that can be

324 easily fermented for the biohydrogen production or other metabolites of interest, containing

325 on average 0.113 g of reducing sugars and 0.0114 g of glucose per gram of vinasses.

326  A maximum biohydrogen production yield of 1.54 (mol H2)/(mol of glucose consumed)

327 was obtained under the following working conditions: initial pH of 3.5, initial substrate

328 concentration of 5 (g glucose)/L and using the Nutrient formulation based on magnesium,

329 iron, zinc and sodium.

330  The results obtained evaluate the potential of mezcal agave vinasses for biohydrogen

331 production.
332

333 Acknowledgements

334 Financial support provided by CIC-UMSNH (Projects 20.20.) is greatly appreciated. ABM and

335 EDAS thank postgraduate studies scholarships from Consejo Nacional de Ciencia y Tecnología

336 (CONACYT). GJG, JAC and RMY greatly appreciate grants from the National System of

337 Researchers (SNI-CONACYT).

338

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