Hydrogen, alcohols and volatile fatty acids from the co-digestion of coffee waste (coffee pulp, husk, and processing wastewater) by applying autochthonous microorganisms

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Hydrogen, alcohols and volatile fatty acids from the co-digestion of coffee waste (coffee pulp, husk, and processing wastewater) by applying autochthonous microorganisms Alejandra Carolina Villa Montoya a,*, Raissa Cristina da Silva Mazareli a, Tiago Palladino Delforno b, Victor Borin Centurion b, Isabel Kimiko Sakamoto a, Val
eria Maia de Oliveira b, Edson Luiz Silva c, ^ ncio Varesche a,** Maria Bernadete Ama ~o Carlos School of Engineering, Laboratory of Biological Processes, Department of Hydraulics and Sanitation, Sa ~o Paulo, Campus II, Sa ~ o Carlos, SP, CEP 13563-120, Brazil University of Sa b Microbial Resources Division, Research Center for Chemistry, Biology and Agriculture (CPQBA), Campinas University, Campinas, SP, CEP 13081-970, Brazil c ~o Carlos, Center of Exact Sciences and Technology, Department of Chemical Engineering, Federal University of Sa ~o Carlos, SP, CEP 13565-905, Brazil Sa a

highlights

graphical abstract


Coffee wastes (CW: wastewater, husk and pulp) for by-products obtainment.
Temperature, pH, headspace and CW

concentration

affected

H2

production.
Maximum values of H2, lactic acid and ethanol were 82 mL, 786 mg/L, 1816 mg/L.
Microbial potential genes related to lignocellulose degradation were explored.
Lactobacillus,

Clostridium

and

Saccharomyces were identified in CW degradation.

article info

abstract

Article history:

The objective of this study was to screen the factors that affect H2, organic acids and al-

Received 1 April 2019

cohols production from coffee waste pretreated in a hydrothermal reactor applying con-

Received in revised form

sortium of bacteria and fungi (indigenous from coffee waste) with hydrolytic and

* Corresponding author. ** Corresponding author. ^ ncio Varesche). E-mail addresses: [email protected] (A.C. Villa Montoya), [email protected] (M.B. Ama https://doi.org/10.1016/j.ijhydene.2019.06.115 0360-3199/© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

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4 June 2019

fermentative activity. The effects of pH (4.0e7.0), temperature (30e50  C), agitation (0

Accepted 19 June 2019

e180 rpm), headspace (50e70%), percentage of bioaugmentation (without microbial con-

Available online 18 July 2019

sortium to 20%), concentration of coffee pulp and husk (2e6 g/L), coffee processing wastewater (7-30 gCOD/L) and yeast extract (0e2 g/L) were evaluated using a Plackett-

Keywords:

Burman design. The highest H2 production potential (82 ml H2) was obtained under the

Ethanol

following conditions: 30  C, 180 rpm, 50% headspace, without bioaugmentation, 2 g/L pulp

Lactic acid

and husk coffee, 30 gCOD/L coffee processing wastewater and 2 g/L yeast extract. The main

Metagenome

soluble products were acetic acid (1956 mg/L), lactic acid (786 mg/L) and ethanol (816 mg/L).

Saccharomyces sp.

Lactobacillus sp., Clostridium sp., Saccharomyces sp. and Kazachstania sp. were the main

Plackett-Burman design

autochthonous microorganisms identified. Through metagenome functional analysis, enzymes related to lignin, phenol, cellulose, lignocellulose, and pectin degradation were identified, as well as acidogenesis, and H2 production. © 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Introduction Agricultural residues represent abundant and interesting raw materials as source of clean energy and bio-products of industrial interest. For instance, during coffee post-harvest processing the generated solid waste (pulp and husk) can reach up to 1 tonne [1] and the amount of wastewater varies between 5000 and 15000 L per ton of coffee [2]. The improper disposal of coffee waste can generate environmental problems such as water eutrophication, acidification and salinization of soils, and toxic effects on some biological processes, aspects which have limited its application in agriculture [3]. The toxicity of coffee waste is due to the presence of polyphenols, compounds that can damage the cell membrane and affect enzymatic activity of the microorganisms [4]. Its concentration can be higher than 9% in coffee pulp and husk and up to 1528 mg/L in the coffee processing wastewater [5,6]. Pulp and husk waste has a high carbohydrate content (70%), of which 16e43% are in the form of cellulose and 7e29% of hemicellulose. Similarly, coffee processing wastewater has a high concentration of organic matter (17244 mgCOD/L), macro and micronutrients (nitrogen, phosphorus and potassium between 23 and 625 mg/L [7,8]). Considering the codigestion of wastewater, pulp and husk, fermentation could increase the availability of substrate and nutritional complexity, leading to greater production of metabolites of interest. The fermentative hydrogen production from coffee drink manufacturing wastewater (31 gCOD/L) was demonstrated by Jung et al. (2012), using UASB reactor at 55  C and 6 h hydraulic retention time (HRT) [9]. The authors observed production of H2 (39.6 LH2/Lsystem/d), ethanol (11%), acetic (13%), caproic (25%) and butyric acids (29%). Samson and Manikkandan (2017) verified for hydrolysed maize stalk (autoclaved with H2SO4 1% for 75 min) 0.91 molH2/mol, during bath reactor operation at pH 7.0, 34.5  C and isolated strain [10]. Asadi and Zilouei (2017) obtained production of 19.73 mlH2/g from pretreated rice straw (180  C, 30 min and ethanol 45% v/v) in bath reactor with Enterobacter aerogenes at pH 5.8 and 37  C [11]. However, no reports were found in the literature for the codigestion of coffee wastes, operational factors,

microorganism performance and important metabolic functions involved in fermentation. One of the main limitations for H2 production from agricultural residues is the low biodegradability of lignocellulosic materials. To overcome these barriers, hydrothermal pretreatment facilitates the decrease of crystallinity and hardness of the lignocellulosic structure, increasing enzymatic access and fermentation yield, and liberating low amounts of inhibitors (such as furfural, 5- hydroxymethylfurfural and phenols) [12]. The application of autochthonous bacteria and fungi (indigenous from coffee waste) with hydrolytic and fermentative activity, also tolerance to high polyphenol concentrations, can overcome the toxicity and recalcitrance of pulp and husk. Bacteria similar to Clostridium sp. have been reported for their ability to degrade several aromatic compounds [13] through cellulolytic and hemicellulolytic enzymes, and for producing H2, ethanol and organic acids [14]. Bacteria similar to Lactobacillus sp. produces lactic acid, in addition to ethanol and acetic acid [15]. These bacteria were identified in UASB reactor producing hydrogen from coffee drink manufacturing wastewater [16]. Similarly, Saccharomyces sp. was observed during the coffee bean fermentation [17], yeasts recognized for their high ethanol production capacity. The fungi can also secrete cellulases, hemicellulases, peptidases, amylases, lignin and phenol degrading enzymes [18], which increase the biodegradability of lignocellulosic waste. Therefore, studies are necessary to make the process economically feasible and to maximize the energy and environmental gain. In this scenario, the application of multifactorial designs can increase the understanding of the most important biological and physicochemical factors for the codigestion of coffee waste. An interesting methodology is the Plackett-Burman design (PB) because it screens a large number of factors with limited experiments, identifying their effect in the process and facilitating the project with a desirable result, which were applied in several studies for H2 production improvement [19]. Through the selection of pH and temperature, specific microbial species, enzymatic activities and metabolic pathways were stimulated [20]. Adequate agitation is important to improve de uniformity and mass transfer efficiency in the reactor [21]. Headspace volume influences the partial pressure

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of hydrogen (pH2), and consequently, the end product formation [22]. Microorganisms or population bioaugmentation with desired catalytic activity can increase substrate degradation for the chosen metabolite [23]. Adequate substrate concentration can augment the community diversity, avoiding microbial competition for carbon and nutrient sources [24]. Yeast extract is a complex nutrient source with nitrogen and micronutrients adequate for microbial growth, which in previous studies have shown to be fundamental for H2 production from lignocellulosic substrates [25,26]. Therefore, the objective of this study was to analyse the effects of initial pH, temperature, headspace volume, agitation speed, percentage of bioaugmentation, concentration of yeast extract, coffee husk and pulp and coffee processing wastewater COD (chemical oxygen demand), in the H2 production from coffee waste with a consortium of autochthonous microorganisms. Additionally, the functional potential and structure of the microbial community was evaluated through metagenomic analyses, providing a comprehensive view of the microbiota associated with fermentation of coffee waste.

Material and methods Obtaining the microbial consortium In order to obtain the microbial consortium, the wet processing of coffee was simulated. One litre of Arabica type coffee beans was placed in 2 L of water for 24 h, and then the pulp and husk were manually removed and dried at 60  C. The resulting liquid (coffee wastewater) was sieved in a 2 mm mesh. Subsequently, successive fermentations were performed, gradually increasing the reactor volume from 250 ml to 2 L after the stabilization of H2 production. The biomass of each reactor was recovered by centrifugation or sedimentation, with resuspension of the pellet in a new medium (coffee processing wastewater, pulp and husk waste and yeast extract). The fermentation conditions were maintained at 35  C, 60% of headspace, 3.5 gCOD/L of wastewater, 2 g/L of pulp and husk waste and 1 g/L of yeast extract. The pH was not adjusted due to the natural acidity of the wastewater (initial pH ~ 4.9). Later, the composition of microbial consortium obtained was studied during analysis of the microbial community through metagenomic sequencing.

Coffee waste Wastewater and coffee pulp and husk waste were collected at ~o Paulo) at the beginning of the “Da lagoa” farm (Pedregulho, Sa the harvest season, after wet processing of the coffee beans. The wastewater was stored in plastic bottles, and frozen at 10  C. The solid waste, which consisted of a mixture of pulp and husk, was oven dried at 60  C in aluminium trays, packed in plastic bags and kept stored at 10  C until use.

Co-digestion of coffee waste The effect of some factors on the fermentation was studied, due to the lack of information on the application of

autochthonous microbial consortium during co-digestion of coffee pulp, husk and wastewater for H2, organic acids and alcohol production. Nath et al. (2011) verified significant effect of pH, temperature, and substrates concentration on H2 production, because they are the most important environmental conditions for microbial growth [27]. However, the improvement of H2 yield through adequate levels of headspace volume [22], agitation [21], bioaugmentation [23] and yeast extract [26] were demonstrated in several previous studies, which motivated us to study these factors. Plackett-Burman design was performed for 8 factors with 3 central points (Table 1) [28]. The factors studied were the initial pH (4.0, 5.5 and 7.0), temperature (30, 40 and 50  C), agitation speed (90, 180 rpm and without agitation), headspace volume (50, 60 and 70%), percentage of bioaugmentation (10%, 20% and without bioaugmentation), pulp and husk concentration (2, 4 and 6 g/L), wastewater concentration (7, 18.5 and 30 gCOD/L), and yeast extract concentration (1, 2 g/L and without yeast extract). The experiments were carried out in batch reactors of 250 ml of useful volume. Microbial consortium from coffee processing waste (0.5 gVSS/L - Volatile Suspended Solids) was used in the bioaugmentation, corresponding to final concentration of 0.05 gVSS/L in reactors with bioaugmentation 10% and 0.1 g VSS/L for bioaugmentation 20%. The microbial consortium was centrifuged at 8000 rpm for 5 min, and the pellet transferred to reactors whose volume is designated in Table 1. Pulp and husk were pretreated in a hydrothermal reactor at 180  C for 15 min (liquid fraction of the pretreatment were not added to the assays). The factors temperature, pH, agitation, headspace volume, percentage of bioaugmentation, concentration of pulp and husk, wastewater and yeast extract were adjusted according the Table 1. The reactors were subjected to N2 (100%) atmosphere for 5 min, the vials were closed with butyl cap and plastic thread.

Physicochemical analysis Carbohydrate, pH, and total suspended solids (TSS) analyses were performed on the samples collected at the beginning and at the end of fermentation [29]. To analyse the solids concentration, the samples were previously subjected to manual or mechanical agitation with glass beads to remove the biomass adhered to the plant material. Filtration in paper filter (7.5 mm) was used to separate the pulp and husk, and filtration in glass fiber membrane (1.2 mm) to retain the microbial biomass.

Chromatographic analyses The quantification of H2 gas was performed by gas chromatography (GC 2010, Shimadzu®) through Carboxen™ 1010 PLOT (30 m  0.53 mm, Supelco) capillary column with argon as carrier gas [30]. Soluble metabolites (organic acids and alcohols) were analysed by gas chromatography (GC 2010, Shimadzu®), using HP-INNOWAX column (30 m  0.25 mm x 0.25 mm) with flame ionization detector (FID), hydrogen as carrier gas, synthetic air and nitrogen as auxiliary gases, and automatic injection (COMBI-PAL sampler AOC5000, Shimadzu®) [30].

2 2 e e e 2 e 2 2 e 2 e 1 1 1 30 7 7 7 30 7 30 30 7 30 30 7 18.5 18.5 18.5 2 2 2 6 2 6 6 2 6 6 6 2 4 4 4

Where: H ¼ Cumulative H2 production (ml H2), P ¼ Maximum H2 production potential Rm ¼ Maximum H2 production rate (ml H2/h), e ¼ 2,718281828, l ¼ Time to start the H2 production (h).

e e 20 e 20 20 e 20 20 20 e e 10 10 10

The effect of the fermentation factors on the kinetics of H2 production, TSS, carbohydrates, organic acids and alcohols were analysed using the software package Minitab version 17.0 with confidence 90%.

50 70 50 70 70 50 70 70 70 50 50 50 60 60 60

Temperature ( C)

30 50 50 30 50 50 50 30 30 30 50 30 40 40 40 7.0 7.0 4.0 4.0 7.0 7.0 4.0 4.0 4.0 7.0 4.0 4.0 5.5 5.5 5.5

() without agitation, bioaugmentation or yeast extract.

1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 1 1 1 1 1 1 1 1 1 1 1 0 0 0

1 1 1 1 1 1 1 1 1 1 1 1 0 0 0

1 1 1 1 1 1 1 1 1 1 1 1 0 0 0

X8 X7 X6 X5

H2),

DNA extraction

X3

X4

(ml

Analysis of the microbial community

X2 X1

(1)

Statistical analysis

180 e 180 180 e 180 180 180 e e e e 90 90 90

Yeast extract (g/L) Wastewater (g./L) Pulp and husk (g/L) Bioaugmentation (%) Headspace (%)

The H2 accumulated production in the headspace was used to perform the H2 production kinetics, adjusting the data to the modified Gompertz model equation (Equation (1)) [31]:    Rm:e ðl  tÞ þ 1 H ¼ P:exp  exp P

pH

Agitation (rpm)

Experimental conditions

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Kinetic analysis



Levels Assay

Table 1 e Plackett-Burman experimental design matrix for evaluating the pH, temperature, headspace, agitation, bioaugmentation, pulp and husk, wastewater and yeast extract concentration in coffee waste co-digestion for hydrogen production.

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At the end of fermentation, the samples were collected and centrifuged at 8000 rpm for 5 min. A composite sample was performed for assays 13, 14 and 15 representing the central point of the PB design (denoted as 13-14-15). These biomasses were washed with PBSX1 buffer (NaCl 8.2 g/L, Na2HPO4 1.05 g/ L, NaH2PO4þH2O 0.35 g/L) and centrifuged again. The pellets were stored at 20  C. To release the microorganisms adhered to the coffee pulp and husk, 3 ml of PBSX1 were added per 1 ml of biomass and glass beads, after which the samples were transfer to Vortex® for 30 s. The glass beads were removed and the liquid was filtered and centrifuged at 8000 rpm for 5 min. The DNA was extracted by FastDNA SPIN Kit for Soil (MPbio) according to the manufacturer's protocol. DNA integrity was verified by 0.8% agarose gel electrophoresis, and DNA quantification and purity analyses were performed on Nanodrop 2000 equipment (ThermoFisher Scientific, https://mlzgarching.de/files/nanodrop_2000_user_manual).

Bioinformatics sequencing and analysis With the DNA samples extracted from the microbial consortium, assays 9 and 13-14-15 the library (short insert DNA e 500 bp) was prepared and the metagenome (3Gbases reads, Paired End 150 bp (Q30 > 85%) was sequenced through the Illumina NovaSeq6000 Platform in the Genone and Biotechnologies laboratory (Rio de Janeiro, Brazil, www.genone. com.br), according to the manufacturer's specifications. The quality of the libraries was checked by the fastqc tool (https://www.bioinformatics.babraham.ac.uk/projects/ fastqc/), and the reads were filtered by the Trimmomatic tool [32] to remove adapters and low quality reads (phred score  20). This was followed by the individual assembly of each library with the metaSPAdes tool [33,34], using k-mers 21, 31, 41, 51, 61, 71, 81, 91, 101. The metaQUAST tool was used to verify the assembly quality [35]. For better annotation, a

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minimum cut of 150 bp was used in the contigs. For the functional, taxonomic annotation and mapping of reads, the FMAP (Functional Mapping and Analysis Pipeline for metagenomic and metatranscriptomic studies [36]) tool was used with 80% identity cutoff, e-value <1e-3 and genes with coverage >80%. FMAP uses its own database for functional annotation, where the UNIREF protein database [37] was filtered by the number of KEGG orthologies (Kyoto Encyclopedia of Genes and Genomes; [38e40]). For the taxonomic annotation, FMAP uses the RefSeq database (NCBI reference sequence database [41]). Sequencing data yielded in this study have been deposited in the European Nucleotide Archive under accession number PRJEB31208.

Results and discussion Kinetic parameters of hydrogen production The effects of the fermentation factors on the maximum H2 production potential (P) and maximum H2 production rate (Rm) were analysed (Fig. S1, Tables 2 and 3). The factor pulp and husk concentration was not significant for H2 production (a ¼ 0.05, Fig. S1). Similar P and Rm were observed for different concentrations of pulp and husk, such as 2 g/L (14 ml H2 and 1 ml H2/h, respectively for assays 1, 2, 3, 5, 8 and 12), 4 g/L (26 ml H2 and 3 ml H2/h, respectively for assays 13, 14 and 15) and 6 g/L (9 ml H2 and 1 ml H2/h, respectively, for assays 4, 6, 7, 9, 10 and 11). In the assays with 6 g/L pulp and husk, long (36 h, assay 6) and short (16.1 h, assay 10) time to start the H2 production (l) were observed. It is likely this was due to the nature of pulp and husk, since the main components of these coffee residues are cellulose and hemicellulose, and therefore their degradation for autochthonous microorganisms by hydrolytic reactions may be incomplete and slow [42]. In agreement with this information, it is possible to infer that a wider range of pulp and husk concentration does not improve the H2 production.

According to Saady et al. (2014), fermentation can be conducted at pH between 4.0 and 7.0, changing the end products formation and growth rate of specific trophic groups [43]. In this research it was interesting to evaluate pH 4.0, because the wastewater collected after wet processing of coffee beans was pH ~3.9. pH was the factor that most affected H2 production (a ¼ 0.1, Fig. S1 and Tables 2 and 3), with the highest P, Rm and l values observed at pH 7.0 (assay 1: 82 ml, 4 ml H2/h and 32.6 h, respectively), intermediate values at pH 5.5 (assay 15: 28 ml, 4 ml H2/h, and 43 h, respectively) and H2 absence at pH 4.0. These results are consistent with those reported by Zhang et al. (2015) from corn stalk, in batch fermentation with Clostridium sartagoforme [44]. The authors evaluated the effect of the initial pH (4.9e8.0) in the fermentation and obtained with higher H2 production (82.7 ml H2/g) in pH 6.47. In pH 4.0 (assays 3, 7, 8, 9, 11 and 12, Tables 2 and 3) no H2 production was observed. Hwang et al. (2004) also reported the absence of H2 production from glucose (5 g/Ld) in a semi-continuously reactor (HRT 3 days) at 35  C and pH < 4.0, due to the inhibition of all microorganism activity [45]. Based on these results it is possible to predict negative effect of pH  4.0 conditions on H2 production. However, according to statistical analyses, substrates with pH  7.0 can lead to higher H2 production, therefore, more studies are suggested regarding this factor. It is important to consider that other pH conditions can stimulate different end products with the autochthonous community, increasing the opportunities of bio-products obtained from coffee wastes. The H2 production evaluated was from 30 to 50  C because the fermentation reactions are possible for mesophilic (2540  C) and thermophilic conditions (40-65  C) [46]. Mesophilic temperatures are especially interesting for coffee waste codigestion, because it can be applied with low operational control in several coffee production areas of countries such as Brazil. The results evidenced that P and Rm decreased on average 44 ml H2 and 2 ml H2/h to 3 ml H2 and 1 ml H2/h, respectively, when the temperature reduced from 50  C (assays 2 and 6, Tables 2 and 3) to 30  C (assay 1, 4 and 10).

Table 2 e Kinetic and physicochemical parameters of co-digestion of coffee waste in bath reactors. Assay

Kinetic parameters P (ml H2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Rm (ml H2/h)

82 3 e 8 3 e e e 40 e e 26 25 28

() Without H2 production.

4 0.1 e 1 e 1 e e e 1 e e 2 3 4

Physicochemical parameters l (h)

32.6 37 e 25.8 e 36 e e e 16.1 e e 42.4 39.7 43

Dissolved carbohydrates

TSS

pH

Initial (mg/L)

Final (mg/L)

Initial (mg/L)

Final (mg/L)

Initial

Final

2617 657 507 613 2667 767 1983 2600 693 1700 2300 420 1267 1267 1267

234 327 479 136 1118 416 2539 639 290 387 2136 268 600 466 566

334 116 112 130 538 112 410 402 174 396 346 98 184 184 184

966 212 115 239 328 322 266 172 250 460 380 153 506 482 320

7.0 7.0 4.0 4.0 7.0 7.0 4.0 4.0 4.0 7.0 4.0 4.0 5.5 5.5 5.5

5.9 5.9 4.2 5.4 5.2 6.1 4.2 3.9 4.2 5.9 4.0 4.1 7.5 6.4 6.6

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Table 3 e Effect of the fermentation factors on carbohydrates reduction, hydrogen, lactic acid and ethanol production, microorganism and enzymes associated. Hydrogen production

pH (X1) Temperature (X2) Agitation (X3) Headspace (X4) Bioaugmentation (X5) Pulp and husk concentration (X6) Wastewater concentration (X7) Yeast extract concentration (X8) Microorganisms Enzymes

Carb. reduction

Lactic acid production

Ethanol production

P

Rm

l

[X1 [P [X2 YP NES [X4 YP NES NES

[X1 [Rm [X2 YRm NES [X4 YRm NES NES

[X1 [l [X2 Yl [ X3 [l [X4 Yl [X5 Yl NES

[X1 [Carb. reduction [X2 YCarb. reduction NES [X4 [Carb. reduction [X5 [Carb. reduction [ X6 YCarb. reduction

NES NES NES NES NES NES

NES NES NES NES NES NES

NES

NES

[X7 Yl

[ X7 [Carb. reduction

NES

NES

NES

NES

[ X8 [l

[X8 [Carb. reduction

NES

NES

Lactobacillus. sp. and Kazachstania sp. Acetaldehyde dehydrogenase/alcohol dehydrogenase and alcohol dehydrogenasepropanol-preferring.

Lactobacillus. sp., Clostridium. sp. and Kazachstania sp. L-lactate dehydrogenase and d D-lactate dehydrogenase.

Clostridium sp. and Saccharomyces sp. Pyruvate-ferredoxin/ flavodoxin oxidoreductase and Formate dehydrogenase

[ Increasing, Y Decreasing, NES Not statistically significant, Carb. Carbohydrates.

Similarly, at 50  C (35.9 h, assays 2, 6 and 11) the l was higher when compared to 30  C (28.4 h, assays 1, 4 and 10). Probably, mesophilic bacteria populations were selected during the preparation of microbial consortium at 37  C [46]. Therefore, it is possible to predict a negative behaviour of autochthonous microorganisms for H2 production in wider fluctuation of temperature, such as thermophilic environments. Nevertheless, additional experimental assays are necessary for other bio-products synthesis. Changes in headspace volume were applied to evaluate the H2 partial pressure (pH2) effect on fermentation, considering that when headspace volume is low in relation to the liquid volume, biogas exerts greater pressure [47]. For H2 production in bath reactor with coffee drink manufacturing wastewater [48] or coffee mucilage and swine manure co-digestion [49] headspace between 24% and 69% was applied. In this study was verified that lower headspace volume favoured a significant increase of P and Rm (a ¼ 0.5, Fig. S1, Tables 2 and 3). Higher values were observed with 50% (P: 82 ml H2 and Rm: 4 ml H2/h in assay 1), followed by 60% (P: 26 ml H2 and Rm: 3 ml H2/h in assays 13, 14 and 15) and finally 70% headspace (P: 3 ml H2 and Rm: 0.1 ml H2/h in assay 2). From these results it can be inferred that lower headspace volume increases the P with the microbial consortium, therefore, a narrower range of headspace is an alternative for optimization of H2 production. According to Villa-Montoya et al. (2016), water recirculation during wet coffee processing is a practice applied to re-use the water and reduce waste generation, conducting to COD from 3 to 50 g/L [6]. Coffee processing wastewater collected from this study showed maximum 31 gCOD/L, therefore, between 10 and 30 gCOD/L were evaluated. Maximum P and Rm of 82 ml H2 and 4 ml H2/h were obtained with 30 gCOD/L of coffee processing wastewater (assay 1). The decrease in these parameters was also noted when the concentration changed from 18.5 gCOD/L (P: 26 ml H2 and Rm: 3 ml H2/h in assays 13, 14 and 15) to 7 gCOD/L (P: 8 ml H2 and Rm: 1 ml H2/h in assay 4).

The l also reduced from 41.7 h (assays 13, 14 and 15) to 16.1 h (assay 10) when the wastewater concentration increased from 18.5 to 30 gCOD/L. This occurred because high wastewater concentration provides greater amounts of dissolved material for the growth of fermentative bacteria, as it contains high concentrations of carbohydrate (2833 mg/L) and organic acids (22110 mg/L). Similar results were observed by Li et al. (2018), evidencing increase in H2 production of 64% with higher soluble COD (from 23.8 to 32.1 g/L) in the substrates, due to the availability of adequate organic matter for fermentation. The authors operated the bath reactor for co-digestion of sewage sludge and food waste at pH 6.1e6.5, 37  C and inoculum from reactor feed with food waste [50]. Therefore, it is possible to conclude that higher wastewater COD can increase the H2 production with autochthonous microorganisms, mainly with coffee wastewater obtained from higher water recirculation. The addition of yeast extract was not required for H2 production from co-digestion of coffee wastes, since the waste themselves may contain vitamins, cofactors and coenzymes. Either in the absence of yeast extract (for example in assay 4, P: 8 ml H2 and Rm: 1 ml H2/h) or with 2 g/L (for example in assay 6, P: 3 ml H2 and Rm: 1 ml H2/h), the P and Rm were close.

Physical-chemical parameters of hydrogen production The physical-chemical parameters were evaluated in order to establish which experimental condition provides greater biomass growth (evaluated by increasing total suspended solids, TSS) and high H2 yields by the substrates consumption (determined by the reduction of dissolved carbohydrates) (Tables 2 and 3, Fig. S2, Supplementary Data). Cell growth and carbohydrate reduction were low or absent in the assays where H2 production was not observed (assays 7 and 3, Table 2). This is because unfavourable operational conditions, such as the acidity of the medium (pH 4.0), thermophilic temperature (50  C), high headspace volume (70%),

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and absence of bioaugmentation, negatively influenced fermentation activity. The maximum carbohydrate reduction of 91% lead to higher P (82 ml H2) and Rm (4 ml H2/L) in assay 1 at pH 7.0, while assay 7 had H2 production absence due to no carbohydrate reduction at pH 4.0 (initial 1983 and final 2530 mg/L). Wang et al. (2015) obtained similar results, reporting glucose increase at pH 4.0 because the low pH promoted hydrolysis and inhibition of acidogenic bacteria. The authors operated bath reactors at 30  C, with food waste as substrate and sludge from UASB reactor of the brewery industry as inoculum [51].

CH3CHOHCOOH(Lactic acid)þ 2H2O /CH3COOH(Acetic  acid) þ HCO3 (Bicarbonate) þ 2H2

Organic acids, alcohols and pH Dissolved metabolites, such as organic acids and alcohols, were analyzed in bath reactors (Fig. 1). Some compounds, such as isobutanol, n-butanol, isobutyric acid, valeric acid, isovaleric acid and caproic acid were observed, but at concentrations below the limit of quantification of the chromatographic method. The effect of initial pH, temperature, agitation, headspace, percentage of bioaugmentation, concentration of pulp and husk, yeast extract and wastewater COD in the productions of acetic, lactic acid and ethanol were studied by means of statistical analyses (Table 3 and Fig. S3, Supplementary Data). The conditions that allowed a significant increase in acetic acid production were neutral pH and mesophilic temperature (a ¼ 0.1). The highest acetic acid production was observed in assay 1 (initial 1111 and final 3197 mg/L) at pH 7.0 and 30  C, while no production was observed in assay 7 (initial 948 to 689 mg/L) at pH 4.0 and temperature of 50  C. This route was favourable for H2 production, since it allowed high H2 yields compared to other metabolic products (Equation (2)), explaining the observed high P of 82 ml in assay 1. C6H12O6(Glucose) þ 2H2O/2 CH3COOH(Acetic (DG ’ ¼ 206)

Similarly, in assays 1, 2 and 6 higher lactic acid concentrations were observed after 20e30 h of operation, but in all cases there was decrease at the end of fermentation. In view of these results, the presence of lactic acid bacteria in the coffee wastewater [48] and the occurrence of the lactic acid route in these reactors (assays 1, 2 and 6) were assumed. However, during the fermentation of coffee waste, the predominant metabolism (lactic pathway) was modified to the acetogenic route [43] (Equation (5)), leading to the conversion of lactic acid to acetic acid and H2 at the end of fermentation.

acid)þ

4H2þ 2 CO2 (2)

The production of enzymatic complexes and catabolic reactions depend on environmental conditions, mainly pH [52]. Thus, it can be considered that pH 7.0 and 30  C were favourable environmental conditions to obtain higher H2 and acetic acid production from the coffee waste. In 13 assays (assays 1, 2, 3, 4, 5, 6, 8, 9, 10, 12, 13, 14 and 15), there was an increase in acetic acid and a decrease in the concentration of other organic acids and alcohols. For example, in assay 10 there was a decrease in ethanol concentration from 2242 mg/L to 1849 mg/L at the end of fermentation. Possibly, the formation of ethanol without H2 production occurred during the first hours of fermentation (Equation (3)), a thermodynamically favourable reaction. However, at the end of the process, the microorganisms transformed the ethanol into acetic acid and H2 through acetogenesis (Equation (4)), due to high concentrations of this acid.

(5)

Mainly in assays with initial pH 4.0, lactic acid was observed at a higher concentration (between 20 and 786 mg/L, assays 3, 5, 9 and 12). The lactic acid production in assays 3, 9 and 12 may be a consequence of the acid pH, since the production of reduced compounds is favoured in this condition, without H2 formation [53]. However, in the statistical analysis, it was verified that none of the factors had a significant effect on the lactic acid production (a ¼ 0.1, Fig. S3, Supplementary Data). The highest lactic acid concentrations of 786 mg/L (in assay 5 at pH 7.0) and 144 mg/L (in assay 9 at pH 4.0) were observed, under different fermentation conditions (Table 1). Lower lactic acid production in assay 9 was consequence of pH 4.0, because only acid tolerant bacteria can ferment at this initial pH. This result was verified by Xiong et al. (2014) during growth of lactic acid bacteria using Chinese sauerkraut at 20e25  C and pH 5.4e5.5 with 4 pure cultures. The authors observed tolerance of just Lactobacillus plantarum and Lactobacillus casei at pH 4.0, with lactic acid production superior to 120 mM [54]. Under the conditions imposed by assay 9 bacteria similar to Lactobacillus sp. was identified with relative abundance of 67.8%. In assay 5, equivalent values of acetic acid, lactic acid and ethanol (0.01, 0.01 and 0.02 mol/L, respectively) were observed, as well as CO2 in the headspace. This result corresponds to the heterolactic fermentation pathway, where hexoses and pentoses are converted into bio-products (such as lactic acid, acetic acid, ethanol, and CO2) by microorganisms such as Leuconostoc sp., Oenococcus sp., and some Lactobacillus sp [55,56]. (Equations (6) and (7)). In assays 9 and 12 there was lactic acid (between 40 and 144 mg/L, Fig. 1) and acetic acid production (between 84 and 375 mg/L) with low CO2 in the biogas. Therefore, the heterolactic fermentation as well as homolactic pathway may have occurred, (Equation (8)), in which the main product is lactic acid from cellobiose without CO2 production [55,56]. C6H12O6(Glucose) / CH3CH(OH)COOH(Lactic acid) þ C2H5OH(Ethanol)þ CO2

(6)

C6H12O6(Glucose) / CH3CH(OH)COOH(Lactic acid) þ CH3COOH (7) (Acetic acid) þ CO2

C6H12O6(Glucose)/CH3CH2OH(Ethanol)þ 2CO2 (DG ’ ¼ 164.8) (3)

C6H12O6(Glucose) / 2CH3CH(OH)COOH(Lactic

CH3CH2OH(Ethanol)þ H2O /CH3COOH (Acetic acid) þ 2H2 (DG ’ ¼ þ 96)

In assay 3 (pH 4.0 and 50  C, Table 1) low production of lactic acid (20 mg/L) and acetic acid was observed (120 mg/L).

(4)

acid)

(8)

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Fig. 1 e Organic acids and alcohols in different phases of coffee waste co-digestion.

Likewise, in assay 7 (pH 4.0 and 50  C), there was no biomass growth (TSS decreased from 410 to 266 mg/L) or carbohydrate reduction (from 1983 to 2539 mg/L). The low and/or no fermentation activity in assays 3 and 7 resulted in the absence of biogas and intermediate compounds in the liquid medium, therefore, these conditions were inhibitory for fermentation and cell growth. Ethanol was only observed in assays 13, 14 and 15 (between 1411 and 1816 mg/L), whose common characteristics were pH 5.5, 40  C, 60% headspace, and others conditions (Table 1), therefore, none of the levels of the factors studied significantly affected ethanol production (a ¼ 0.1, Fig. S3, Supplementary Data). Dionisi and Silva (2016) affirmed that ethanol production is favoured by pH higher than 6.0. In the specific case of batch reactors with coffee waste, pH of 7.5, 6.4 and 6.6 (assay 13, 14 and 15, respectively) were found at the end of fermentation in these reactors, explaining high concentrations of ethanol in these assays (Equation (9)). In equation (9) there is the bicarbonate formation, which justifies the increase in pH value from 5.5 to 7.5, 6.4 and 6.6 at the end of fermentation in assays 13, 14 and 15, respectively. Alkalinity contribution by the conversion of lactic acid to acetic acid (Equation (3)) can also be considered, since a decrease in lactic acid concentration was observed in assays

13, 14 and 15 (~5268 mg/L at the beginning and 183 mg/L at the end of fermentation). C6H12O6(Glucose) þ 2H2O þ 2NADH þ /2CH3CH2OH(Ethanol) þ 2HCO 3 (Bicarbonate) þ 2NAD þ 2H2

(9)

There was an increase in methanol concentration in assay 10 (from 107 to 341 mg/L) and assays 13, 14 and 15 (from 102 to 230 mg/L). Schink and Zeikus (1980) observed that methanol can be produced during the anaerobic digestion of fruits and vegetables from the pectin contained in these wastes by pectinolytic enzymes from bacteria such as Clostridium sp. or Pseudomonas sp.

Metagenome -taxonomic profile Samples from the microbial consortium, assays 9 and 13-1415 (representing the central point of the PB design) were sequenced in order to identify differences in the microbial community and functional genes for autochthonous microorganisms from coffee waste, in assays where lactic acid (assay 9) and ethanol (assays 13-14-15) were observed. The rarefaction curves of the taxonomic data were analyzed in the Supplementary Data (Figs. S4 and S5) [57].

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The relative abundance of 94.7%, 95% and 91.6% related to the Bacteria domain was verified through taxonomic evaluation (Figs. 2 and 3), 0.01%, 1.2% and 0.87% related to the Eukaryote domain and maximum 0.17% related to the Archaea domain for the microbial consortium, assays 9 and 13-14-15, respectively. In all assays a similar bacterial community (~92%) demonstrated the ability to ferment various substrates and obtain different products according to environmental conditions. For the Eukaryotic domain, the fungal population represented 0.092%, 0.008% and 0.003% of relative abundance in the microbial consortium, assays 9 and 13-14-15, respectively. The decrease in the fungal population from the microbial consortium (3.5 gCOD/L) in assays 9 and 13-14-15 (7 gCOD/L and 18.5 gCOD/L, respectively) may be related with higher coffee wastewater concentration during fermentation. According to Nagpal et al. (2009) fungi population in anaerobic environments, such as rumen, is low during diet rich in soluble carbohydrates and high during diet rich in lignocellulosic substrate [58], explaining low fungal population in assays 9 and 13-14-15 with dissolved carbohydrates higher than 693 mg/L. The Archaea relative abundance of 0.17% in the microbial consortium may have been a consequence of their presence in the substrates, since CH4 was not detected in this reactor. Bacteria from the family Clostridiaceae (89.2%) were the most abundant in the microbial consortium, Lactobacillaceae (68.2%) and Acetobacteraceae (16.1%) in assay 9 and Clostridiaceae (34.6%), Lactobacillaceae (33.6%) and Acetobacteraceae in assays 13-14-15 (Fig. 2 and Table A, Supplementary Data). Clostridiaceae was referred to as the main family related to the H2 production in anaerobic reactors [59]. On the other hand, family Lactobacillaceae was characterized by its wide fermentative capacity, with production of lactic acid, ethanol, CO2, formic acid and succinic acid from carbohydrates [60]. Lactobacillaceae has growth capacity at pH 4.0 [61], explaining the high relative abundance observed for representatives of this family in assay 9. The bacteria from the family Acetobacteraceae are commonly identified in acidic environments such as fruits [62], which may have originated from the coffee pulp and husk. Clostridium sp., Lactobacillus sp. and Acetobacter sp. were the major bacterial genera identified in all assays (Fig. 2 and Table A, Supplementary Data). A relative abundance of 76.9% of Clostridium sp. was observed in the microbial consortium, a genus referenced for its capacity to ferment sugars for butyric acid production, and degrade cellulose for the production of alcohols and organic acids [14]. The relative abundance of Lactobacillus sp. increased from 2.9% in the microbial consortium to 67.8% in assay 9. Representatives of this genus are acid tolerant and have fermentative metabolism at acidic pH of 3.5 [55], similar conditions to those observed during assay 9 (initial pH 4.0 and 2199 mg/L of lactic acid). The population of Acetobacter sp. of 15.5% was significant in assay 9, however, bacteria of this genus were characterized for having aerobic metabolism [63]. Joyeux et al. (1984) studied the growth of acetic acid bacteria during the different stages of wine production, and determined that Acetobacter pasteurianus and A. aceti can survive aerobic and anaerobic processes, because they grow during short exposure of wine to air [64]. The results of these authors may explain the identification of bacteria of the

genus Acetobacter sp. in samples collected from reactors under anaerobic conditions. Other genera identified in assay 9 were Paenibacillus sp. (3.2%), Anoxybacillus sp. (2.4%) and Bacillus sp. (2.2%) of the family Bacillacea. Bacteria related to this family are usually detected in fermented foods [65], therefore, originating from coffee waste. The higher relative abundance in assay 9 may be a consequence of its ability to form endospores [65], which allow them to survive to the restrictive environmental conditions set in this assay. In the samples from assays 13-14-15, Lactobacillus sp. (33.4%), Clostridium sp. (22.6%) and Acetobacter sp. (13.4%) cohabited as a consequence of the neutral pH observed at the end of fermentation (between 6.4 and 7.5), suitable for the growth of several bacterial genera [66]. Hungatella sp. (3.3%) was identified in these assays due to optimum growth at pH 7.0 and 30  C [67], conditions similar to those observed at the end of these assays (neutral pH and 40  C). In addition, Hungatella sp. produces acetic acid, ethanol, H2 and CO2 from glucose [67], metabolites observed at the end of fermentation in assays 13, 14 and 15 (Fig. 1). Lachnoclostridium sp. was possibly favoured in assays 13-14-15 (3.2%) due to the preference for mesophilic and neutral pH environments, and their ability to ferment mono and disaccharides for acetic acid production [68], whose observed concentration was between 1062 and 1307 mg/L. Low relative abundance for fungi was verified in the different assays (Fig. 3), with a predominance of Ascomycota of 99.2% in the microbial consortium, 98.1% in assay 9 and 100% in assays 13-14-15. Ascomycota is characterized by its ability to degrade plant material through enzymes such as cellulases and xylanases [69], important for the hydrolysis of coffee pulp and husk. Relative abundances between 0.1% and 15.5% were observed for the different fungal genera in the microbial consortium, in assays 9 and 13-14-15. The fungi genera identified were Saccharomyces sp. (11.4e15.2%), Kazachstania sp. (13.8e15.2%) and Lachancea sp. (4.9e8.6%). Similar relative abundance of fungi in the different assays allowed inferring that the fermentative conditions did not influence the growth of specific genera. Therefore, the same origin of substrates and bioaugmentation with the microbial consortium was more important for the conformation of the fungal community. Saccharomyces sp. was identified in coffee beans for the removal of pulp and mucilaginous material, with growth between 20 and 45  C, pH between 2.5 and 8.0 and high ethanol concentration [70], adapting to the different conditions set in the assays. Saccharomyces sp. may be important when the goal is alcohol production because of its ability to convert carbohydrates into ethanol with high final concentrations [70]. Kazachstania sp. can ferment glucose, galactose and sucrose from materials such as fruits, adapting to temperatures between 25 and 40  C and tolerating acetic acid, conditions observed in assays 13-14-15 (between 1599 and 1843 mg/L), which explains its predominance in this assay (15.2%). Greater relative abundance of Lachancea sp. was observed in assays 13-14-15 due to the high ethanol concentration, as this yeast can produce lactic acid and reduce volatile acids in alcoholic substrates such as wine [71], explaining the lower concentrations of volatile acids at the end of fermentation in assays 13, 14 and 15 (1892 mg/L).

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Fig. 2 e Taxonomic groups for Bacteria (a) Families and (b) Genus from coffee waste co-digestion. *Relative abundance calculated considering the total reads in the samples from microbial consortium (2297166), assay 9 (10776878) and assays 13-14-15 (5562693).

Kazachstania sp. and Lachancea sp. may use some organic acids produced by lactic acid bacteria [72], justifying their higher abundance in assays 9 and 13-14-15, where relative abundance of these groups of bacteria (Lactobacillus sp.) were higher (Fig. 2 and Table A, Supplementary Data).

In other studies, microorganisms such as Bacillus subtilis, Bacillus cereus, and Pichia anomala were identified during semi-dry coffee processing [73], therefore, coffee wastes were an important source of microorganisms with fermentative and hydrolytic metabolisms.

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Fig. 3 e Taxonomic groups for Fungus (a) Phylum and (b) Genus from coffee waste co-digestion. *Relative abundance calculated considering the total reads in the samples from microbial consortium (2297166), assay 9 (10776878) and assays 13-14-15 (5562693).

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According to the taxonomic analyses, high microbial diversity was verified in the coffee wastes. The operational conditions were compatible to the growth of specific genus from autochthonous microorganisms. For example, fermentation conditions such as pH 4.9, 37  C, 2 g/L pulp and husk, and 3.5 gCOD/L wastewater stimulated the activity of Clostridium sp. (microbial consortium) with H2 production. On the other hand, under conditions of pH 4.0, 30  C, 6 g/L pulp and husk, and 7 gCOD/L wastewater Lactobacillus sp. (assay 9) and lactic acid production predominated. In the assay operated at pH 5.5, 40  C, 4 g/L pulp and husk, and 18.5 gCOD/L wastewater, the association between Lactobacillus sp. and Clostridium sp. was observed, with ethanol and acetic acid production (assays 13-14-15). Therefore, the fermentation factors such as temperature, pH and substrate concentration changed the microbial community structure. The possibility to stimulate a specific genus from the autochthonous microorganisms of coffee wastes were similar to that observed in Rabelo et al. (2018), with 23 mmol H2/L from sugarcane bagasse with autochthonous cellulolytic bacteria in bath reactors at pH 7.2 and 37  C [74]. In contrast, Soares et al. (2018) observed H2 consumption by methanogenic archaea in bath reactor with sugarcane bagasse at pH 6.0 and 55  C. Inhibition strategies for thermophilic sludge from an UASB reactor were not enough for avoid unwanted metabolisms in the reactors [75].

Metagenome - functional profile Potential functional profile in the microbial consortium, assays 9 and 13-14-15, has been studied through KO (KEGG Orthology) annotation according to the KEGG database (Kyoto Encyclopedia of Genes and Genomes), which represent genes/ proteins with specific functions. Rarefaction curves built with KO numbers (Fig. S6) and Kegg category Level 2 composition were discussed in the Supplementary Data (Fig. S7) [75]. Functional analyses were focused on metabolisms related with degradation of lignin and phenol, pectin, cellulose and hemicellulose, further acidogenesis, acetogenesis and fungal enzymes (Fig. 4 and Fig. S8, Supplementary Data). These metabolisms correspond to 4.6%, 3.5 and 3.6% of total KOs determined in the DNA samples from the microbial consortium, assays 9 and 13-14-15, respectively. The microbial consortium was the sample with the highest relative abundance related to metabolism relevant for coffee waste biodegradation, likely due to the bioaugmentation process. According to Sivagurunathan et al. (2016), bioaugmentation allows the selection of specialized catalyst, explaining the increase in enzymes that act in lignocellulose, pectin, phenols and carbohydrates, common compounds found in coffee waste [76]. By means of the comprehensive study of these metabolisms, the following was observed: 7% for lignin and phenol degradation, 37.9% for cellulose and hemicellulose degradation, 33.7% for acidogenesis, 0.1% for acetogenesis, 12.7% for hydrogen production and 8.6% for fungal enzymes in the microbial consortium (Fig. 4 and Fig. S8). These enzymes were important in coffee waste degradation due to its participation in the hydrolysis of polymers, carbohydrates conversion into organic acids and biogas, and the production of less toxic effluent due to phenol degradation. Moreover, enzymes from

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fungus such as cellulases, hemicellulases, and ligninases [77], contribute with additional hydrolysis of pulp and husk. In the microbial consortium, the most important metabolism was cellulose and hemicellulose degradation, where K01223 (3%) and K01811 (3.3%) corresponded to 6-phosphobeta-glucosidase (E.C: 3.2.1.86) and alpha-D-xyloside xylohydrolase (E.C: 3.2.1.177), respectively. Both enzymes are from the family glycoside hydrolase, the first acts over carbohydrates producing glucose and other monosaccharides, which have been found in Escherichia coli and Lactobacillus lactis [78]. The second, alpha-D-xyloside xylohydrolase from bacteria such as Clostridium cellulovorans, metabolizes galactomannan of hemicellulose [79]. The second predominant metabolism in the microbial consortium was acidogenesis (33.7%). The K00016 (L-lactate dehydrogenase, EC: 1.1.1.27) and K04072 (acetaldehyde dehydrogenase/alcohol dehydrogenase, EC: 1.2.1.10) showed relative abundances of 2.5% and 4.7%, respectively. The L-lactate dehydrogenase transforms pyruvate into lactic acid during heterolactic fermentation [55], and acetaldehyde dehydrogenase synthesizes acetaldehyde in the previous reaction to ethanol generation during solvent-producing clostridia [80]. These findings can explain the origin of metabolism development in assay 9 with lactic acid production of 144 mg/L, and in assays 13-14-15 with ethanol production of ~1602 mg/L. Therefore, it is important to consider the presence of butyrate kinase (2.5%, K00929, E.C: 2.7.2.7) and acetate kinase (1%, K00925, E.C: 2.7.2.1) because these enzymes produce butyric acid and acetic acid from lactic acid [15], as observed in the microbial consortium. Fungal enzymes contributed 8.6% to the microbial consortium metabolism. The K01958 and K00873 showed 4% and 3.5% of relative abundance, respectively, corresponding to pyruvate carboxylase (E.C: 6.4.1.1) and pyruvate kinase (E.C: 2.7.1.40), important enzymes for the growth of Saccharomyces cerevisiae from carbohydrates [81,82]. Other interesting enzymes were alcohol dehydrogenase (K13953, E.C: 1.1.1.1) and aldehyde dehydrogenase (K00128, E.C: 1.2.1.3) with relative abundance of 0.1% and 0.5%, respectively, related to alcoholic fermentation and identified in several yeasts [55,83]. This diversity of fungal enzymes revealed their important contribution to ethanol production from coffee waste. Potential for H2 production with 12.7% of relative abundance has been related to pyruvate-ferredoxin/flavodoxin oxidoreductase (11%, K03737, E.C: 1.2.7.1 1.2.7.-) and pyruvate ferredoxin oxidoreductase (E.C: 1.2.7.1, 0.3% for alpha subunit - K00169; 0.3% for beta subunit - K00170, 0.1% for delta subunit - K00171 and 0.1% for gamma subunit - K00172). These enzymes break down the pyruvate forming intermediaries for H2 production in clostridial-type fermentation [84]. Predominance of Clostridium sp. in the microbial consortium (76.9%) explained that 10.4% of metabolism for coffee waste degradation was related to H2 production. Ferredoxin hydrogenase of 0.1% (K00532, E.C: 1.12.7.2), formate dehydrogenase of 0.03% (K00122, E.C: 1.17.1.9) and formate dehydrogenase major subunit of 0.7% (K00123, E.C: 1.17.1.9) were also found in the microbial consortium. Ferredoxin hydrogenase allows pyruvate conversion into acetic acid, CO2, and H2 by anaerobic fungus, due to a special organelle called hydrogenosome [84]. The last two enzymes

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are from the group of formate dehydrogenases, which have been found in yeast and aerobic and anaerobic bacteria for formic acid oxidation. In anaerobic conditions, some bacteria transform the formic acid into H2 and CO2 through the enzyme formic hydrogenlyase [85]. The most important metabolism in assay 9 was acidogenesis with 42.4%, which has been annotated as 6% of K00016 (L-lactate dehydrogenase, E.C: 1.1.1.27) and as 5.3% of K04072 (acetaldehyde dehydrogenase/alcohol dehydrogenase E.C: 1.2.1.10 1.1.1.1). The 67.8% of Lactobacillus sp. in this assay explains the predominance of lactic acid metabolism, with a production of 144 mg/L. On the other hand, the acetaldehyde dehydrogenase/alcohol dehydrogenase was related to the transformation of acetaldehyde into ethanol during enterictype mixed-acid fermentation [84], however, ethanol was not produced. Degradation of cellulose and hemicellulose had 34.1% of relative abundance, with 4.5% for 6-phospho-beta-glucosidase

(K01223; E.C: 3.2.1.86), 4.1% for alpha-galactosidase (K07407, E.C: 3.2.1.22) and 3.9% for beta-galactosidase (K01190, E.C: 3.2.1.23). These galactosidases were relevant to pulp and husk rupture because they act over mannan and hetero-mannans of lignocellulose to produce galactose residues [86], which is adequate material for fermentation. In addition, enzymes required to degrade polymeric material from pectin into monosaccharides were relevant in assay 9, with relative abundance of 0.03% for pectate lyase (K01728, E.C: 4.2.2.2), 0.03% for rhamnogalacturonan exolyase (K18198, E.C: 4.2.2.24) and 0.03% for rhamnogalacturonan endolyase (K18197, E.C: 4.2.2.23) [57]. Subsequently, fungal enzymes in assay 9 corresponded to relative abundance of 9.9%, such as pyruvate kinase with 3.9% (E.C: 2.7.1.40, K00873), alcohol dehydrogenase with 3% (E.C: 1.1.1.1, K13953) and pyruvate carboxylase with 1.9% (EC:3.2.1.26, K01193). These enzymes were related to carbohydrate fermentation and ethanol pathway in yeast, however,

Pulp, husk and wastewater Pectin

Polyphenol

Lignocellulose

Bacillus: 0%/0,002%/0

4.2.2.2/4.2.2.24/4.2.2.23

Lignin/phenol Clostridium: 0,128%/0,004%/0,013% Lactobacillus: 0,005%/0,168%/0,053%

Hemicellulose

Cellulose

Clostridium: 0,498%/0,007%/0,055% Lactobacillus: 0,015%/0,153%/0,117%

2.3.1.9/1.1.1.90

3.2.1.4//3.2.1.21/ 3.2.1.86/3.2.1.70

Galactose

Mannose

Arabinose 3.2.1.22/3.2.1.23 /3.2.1.85

Clostridium: 0,002%/0%/0% Paenibacillus: 0%/0,001%/0% Bacillus: 0%/0,001%/0%

3.2.1.55/3.2.1.99

5.3.1.8/5.2.1.25/ 2.7.7.13/5.4.2.8

Xylose Clostridium: 0,170%/0,001%/0,005% Paenibacillus: 0,015%/0,014%/0% Lactobacillus: 0,001%/ 0,022%/0,006%

Clostridium: 0,153%/0,002%/0,010% Lactobacillus: 0,011%/0,288%/0,137% Clostridium: 0,090%/0,003%/0,010% Paenibacillus: 0%/0,007%/0% Lactobacillus: 0,002%/0,048%/0,026% Bacillus: 0%/0,002%/0% Paenibacillus: 0%/0,001%/0%

3.2.1.8/3.2.1.37/ 3.2.1.177/3.2.1.156

Monosaccharides

1.2.7.1 1.2.7 Clostridium: 0,413%/0,079%

H2

1.2.1.10 1.1.1.1

Clostridium: 0,089%/0,012% Lactobacillus: 0,004%/0,057% Saccharomyces: 0,0003%

Ethanol

2.7.2.1

Clostridium: 0,034%/0,001%/0,038% Lactobacillus: 0,006%/0,098%/0,052%

1.1.1.27

Organic acids/Alcohols

Lactobacillus: 0,005%/0,188%

Acetic acid Lactic acid

Fig. 4 e Proposed metabolic pathways for H2, organic acids and alcohols production in co-digestion of coffee waste under different operational conditions. Relative abundance* for enzymes in Microbial consortium (Blue number and /, pH 4.9, 37  C, without agitation, 60% headspace, 2 g/L pulp and husk, 3.5 gCOD/L wastewater, 1 g/L yeast extract), assay 9 (Green number and /, pH 4.0, 30  C, without agitation, 70% headspace, 20% bioaugmentation, 6 g/L pulp and husk, 7 gCOD/L wastewater, 2 g/L yeast extract), and assays 13-14-15 (Red number and /, pH 5.5, 40  C, 90 rpm, 60% headspace, 10% bioaugmentation, 4 g/L pulp and husk, 18.5 gCOD/L wastewater, 1 g/L yeast extract). Enzymes (EC): 4.2.2.2) Pectate lyase, 4.2.2.23) Rhamnogalacturonan endolyase, 4.2.2.24) Rhamnogalacturonan exolyase, 2.3.1.9) Acetyl-CoA C-acetyltransferase, 1.1.1.90) Aryl-alcohol dehydrogenase, 3.2.1.4) Endoglucanase, 3.2.1.21) Beta-glucosidase, 3.2.1.86) 6-phospho-betaglucosidase, 3.2.1.70) Glucan 1,6-alpha-glucosidase, 3.2.1.55) Arabinoxylan arabinofuranohydrolase, 3.2.1.99) Arabinan endo-1,5-alpha-L-arabinosidase, 3.2.1.22) Alpha-galactosidase, 3.2.1.23) Beta-galactosidase, 3.2.1.85) 6-phospho-betagalactosidase, 5.3.1.8) Mannose-6-phosphate isomerase, 3.2.1.25) Beta-mannosidase, 2.7.7.13) Mannose-1-phosphate guanylyltransferase, 5.4.2.8) Phosphomannomutase, 3.2.1.8) Endo-1,4-beta-xylanase, 3.2.1.37) Xylan 1,4-beta-xylosidase, 3.2.1.177) Alpha-D-xyloside xylohydrolase, 3.2.1.156) Oligosaccharide reducing-end xylanase, 1.2.7.1 1.2.7.-) Pyruvateferredoxin/flavodoxin oxidoreductase, 1.2.1.10 1.1.1.1) Acetaldehyde dehydrogenase/alcohol dehydrogenase, 2.7.2.1) Acetate kinase, 1.1.1.27) L-lactate dehydrogenase.*Relative abundance calculated considering the total reads in the samples from microbial consortium (2297166), assay 9 (10776878) and assays 13-14-15 (5562693).

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ethanol production was not observed in this assay. Probably, alcohol dehydrogenase was present during metagenome sequencing, but not active during fermentation. In assay 9 the highest relative abundance of 9.2% for lignin and phenol degradation was observed, in comparison to the microbial consortium (7%) and assays 13-14-15 (5.6%), with important enzymes such as aryl-alcohol dehydrogenase (0.9%, EC: 1.1.1.90, K00055) and acetyl-CoA C-acetyltransferase (1.4%, EC: 2.3.1.9, K00626). Wu et al. (2019) reported negative effect of enrichment of H2 producers on the reactor, due to an increase of the genus associated with acids production (representatives of Firmicutes, from 10% to 16%) and decrease of phenol degraders (representatives of Proteobacteria, from 39% to 34%). In assay 9 higher relative abundance for the phenol degrading genus was observed, similar to that observed by the authors [87]. The aryl-alcohol dehydrogenase reduces aromatic compounds into low molecular mass intermediates during lignin degradation [88]. The acetyl-CoA C-acetyltransferase has been found in Clostridium acetobutylicum for the transformation of aromatic compounds through benzoate degradation with coenzyme A ligases. This enzyme transforms acetyl-CoA into acetoacetyl-CoA, which can be converted into organic acids and alcohols [89]. Under operational conditions of assay 9 (Table 1), the KOs were related to hydrolytic reactions from bacteria and fungus. This explains the higher diversity and richness of the genus in assay 9 without H2 production, corresponding to biomass growth (from 174 to 250 mg TSS/L) and carbohydrate reduction (75%) determined at the end of fermentation. In assays 13-14-15, KO numbers from Fig. S8 represented 3.6% of the sample. Greater acidogenesis activity was found in this assay (44.9%), contributing to higher ethanol and acetic acid concentration observed. Enzymes associated with these results were acetaldehyde dehydrogenase/alcohol dehydrogenase (K04072, E.C: 1.2.1.10 1.1.1.1) and acetate kinase (K00925, E.C: 2.7.2.1), with relative abundance of 6.9 and 2.9%, respectively. Degradation of cellulose and hemicellulose represented 31.8% of KOs in assays 13-14-15 (Fig. S8). The most abundant enzyme was 6-phospho-beta-glucosidase (K01223, E.C: 3.2.1.86) with 5.8%, due to 87.9% predominance of Clostridium sp. in this assay, bacteria with hydrolytic and fermentative capacity that explains the co-production of H2, ethanol, and acetic acid [78]. Moreover, fungal enzymes in assays 13-14-15 represented 8.2% of KOs in Fig. S8. The most abundant enzymes were pyruvate kinase with 3.9% (E.C: 2.7.1.40, K00873) and pyruvate carboxylase with 1.7% (E.C: 6.4.1.1, K01958). The last enzyme relative abundance decreased when compared with the microbial consortium (4%). This result was a consequence of lower fungal diversity in this assay, and higher bacteria activity with ethanol production capacity. Fig. 4 shows a proposed route for production of H2, organic acids and alcohols from coffee waste (pulp, husk and wastewater), considering possible substrates found in the waste, metabolic products quantified in the reactors, enzymes and microorganisms with greater relative abundance during metagenomic analysis. Enzymes with potential for transformation of all compounds were found in the coffee wastes

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and production of metabolic products quantified at the end of fermentation in assays 9 and 13-14-15 (H2, ethanol, lactic acid and acetic acid). Clostridium sp. was the most important microorganism for the co-digestion of coffee waste, identifying greater relative abundance in reactions for hydrolysis of lignin/phenol (0.004%e0.128%), cellulose and hemicellulose (up to 0.408%), production of H2 (0.079%e0.413%), ethanol (0.012%e0.089%) and acetic acid (0.001%e0.034%). Lactobacillus sp. was the second microorganism with the highest metabolic potential; however, it was limited to the production of ethanol (0.004%e0.057%), acetic acid (0.006%e0.098%), and lactic acid (0.005%e0.188%). Bacillus sp. was important for the degradation of pectin (0%e0.002%), arabinose (up to 0.001%) and galactose (up to 0.002%), and Paenibacillus sp. of all hemicellulose sub-components (up to 0.014%). Therefore, autochthonous microorganisms from the pulp, husk and wastewater had a high metabolic potential for a wide variety of transformation routes of coffee waste into acids, alcohols and H2. On the other hand, most of the enzymes were observed in all analyzed samples (microbial consortium, assays 9 and 1314-15) for the degradation of polyphenols and lignocellulose, observing differences during the transformation of monosaccharides into gaseous or dissolved metabolites. Similarly, pectin degradation was only verified in assay 9 (0.002%), therefore the conditions of this assay (Table 1) stimulated higher growth of microorganisms with this metabolism, similar to Bacillus sp.

Conclusions
Bacteria and fungus were identified in coffee waste, with functional genes related to hydrolytic and fermentative metabolism and delignification capacity under several operational conditions.
The main physical-chemical factors enhancing H2 production were pH 7.0, 30  C, 50% headspace and high substrate concentration (30 gCOD/L wastewater, 6 g/L pulp and husk).
Lactic acid or ethanol production from coffee waste is possible by the appropriate selection of fermentation factors, such as lactic acid at pH 4.0 and 30  C and ethanol at pH 5.5 and 40  C.
Predominance of Clostridium sp., Lactobacillus sp., Saccharomyces sp. and Kazachstania sp. in the reactors with metabolisms related to degradation of lignin, phenol, cellulose, hemicellulose, and pectin, in addition to acidogenesis, acetogenesis, and hydrogen production.
Higher relative abundance of enzymes related to degradation of cellulose and hemicellulose were observed in the microbial consortium (such as the 6phospho-beta-glucosidase) and with acidogenesis in assays 9 (such as the L-lactate dehydrogenase) and 1314-15 (such as the acetaldehyde dehydrogenase/alcohol dehydrogenase).
The potential of coffee waste and autochthonous microorganisms for energy recovery (such as H2), alcohols (such

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as ethanol), and organic acids (such as lactic, and acetic acid) production with industrial applications.
Co-digestion of coffee pulp, husk and wastewater represents higher waste exploitation in coffee farms. In addition, adequate fermentation conditions without nutrients addition (like yeast extract), agitation, and high temperature control were verified, which represent possible cost reductions. However, more studies are necessary to optimize the gaseous and liquid metabolite production and apply this technology in a pilot or industrial scale in continuous reactors.

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Acknowledgments ~ o de The authors thank the financial support of the Fundac¸a  Pesquisa do Estado de Sa ~ o Paulo (FAPESP- Process Amparo a ~ o de number 2016/20047-0 and 2015/06246-7) and Coordenac¸a Aperfeic¸oamento de Pessoal de Nı´vel Superior - Brasil (CAPES) - Finance Code 001. We also thank the ‘‘Da Lagoa’’ farm ~ o Paulo, Brazil) to provide the coffee waste. (Pedregulho, Sa

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Appendix A. Supplementary data [15]

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijhydene.2019.06.115.

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