Production of 1,3-propanediol from pure and crude glycerol using a UASB reactor with attached biomass in silicone support

Accepted Manuscript Production of 1,3-propanediol from pure and crude glycerol using a UASB reactor with attached biomass in silicone support S.T.S. Veras, P. Rojas, L. Florencio, M.T. Kato, J.L. Sanz Martín PII: DOI: Reference:

S0960-8524(19)30158-0 https://doi.org/10.1016/j.biortech.2019.01.125 BITE 21002

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

28 November 2018 24 January 2019 25 January 2019

Please cite this article as: Veras, S.T.S., Rojas, P., Florencio, L., Kato, M.T., Sanz Martín, J.L., Production of 1,3propanediol from pure and crude glycerol using a UASB reactor with attached biomass in silicone support, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.01.125

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1 Production of 1,3-propanediol from pure and crude glycerol using a UASB reactor with attached biomass in silicone support

S. T. S. Verasa,b, P. Rojasa, L. Florenciob, M. T. Katob, J. L. Sanz Martína,*

a

Universidad Autónoma de Madrid, Department of Molecular Biology, Madrid 28049,

Spain. E-mail: [email protected], [email protected], [email protected] b

Federal University of Pernambuco, Department of Civil and Environmental

Engineering, Laboratory of Environmental Sanitation, Recife PE, 50740-530, Brazil. Email: [email protected], [email protected]

*Corresponding author: J. L. Sanz Martín E-mail: [email protected] Darwin 2, Universidad Autónoma de Madrid, 28049 Madrid, Spain. Phone: +34 914974303.

2 Abstract The 1,3-propanediol (1,3-PDO) yield and productivity from glycerol were studied over a 155-day period. A UASB reactor that also contained silicone support for biomass attachment was used to evaluate the optimal operational conditions and microbiota development. The highest average 1,3-PDO yield was 0.54 and 0.48 mol.mol-gly-1 when reactor pH was 5.0-5.5 and the applied loading rate was 18 and 20 g gly.L-1.d-1 using the pure and crude substrate, respectively. The productivity was close to 7.5 g.L1

.d-1 for both substrates; therefore, the direct use of crude glycerol can be valorized in

practice. Clostridium was the predominant genus for 1,3-PDO production and C. pasteurianum was dominant in the biofilm. Using crude glycerol, C. beijerinckii dropped strongly some Clostridium population was then replaced by Klebsiella pneumoniae and Lactobacillus spp. The good process performance and the advances in the microbiota knowledge are steps forward to obtain a more cost-effective system in practice.

Keywords: glycerol; 1,3-propanediol; mixed microbial consortia; UASB; Illumina; Clostridium

3 1. Introduction 1,3-propanediol (1,3-PDO) is broadly used in the production and formulation of many products, i.e. (i) in polymer production, such as polytrimethylene terephthalate (PTT), which presents advantages and a competitive price in relation to polyethylene terephthalate (PET) and butylene polyterephthalate (PBT) (Zeng and Sabra, 2011); (ii) as additives in the food and pharmaceutical industries; (iii) as additives to improve solvent properties; (iv) in the production of adhesives, polyester resins, inks, lubricants, antifreezes, cosmetics, and biocides (Saxena et al., 2009; Sun et al., 2018). Due its widespread use, improving its production is both a challenge and a valuable contribution, mainly from an economic standpoint. More than one hundred thousand tons of 1,3-PDO (1.8 €/ kg market price, Burniol-Figols et al., 2018) are produced annually and most come from sugars or glycerol by fermentation processes (E4tech et al., 2015). These processes substituted the chemical synthesis because of the expensive catalysts cost, and the production of toxic intermediates. Biological processes have been able to overcome these disadvantages (Nakazawa et al., 2017; Yang et al., 2017). Moreover, they are more environmental friendly than the chemical processes because of the use of renewable waste material like glycerol as substrate. The first time 1,3-PDO was synthesized from glycerol occurred in 1881 (Werkman and Gillen, 1932). But its increasing production can firstly be attributed to the marketing strategies used by DuPont in 1995 and Shell in 1998, for the use of PTT. More recently, due to the exponential increase of the global biodiesel production estimated at over 42 million m3 until 2026 (OECD/FAO, 2017), more than approximately 4 million m3 of glycerol can be generated and therefore, be used for the production of 1,3-PDO and other value-added compounds. Biodiesel production occurs via a transesterification reaction between a fat or oil

4 and an alcohol in the presence of a catalyst, where approximately 10 % (w/w) corresponds to the glycerol formed (Silva et al., 2009). Glycerol is used in the production of cosmetics, pharmaceuticals, foods and fuel additives, surfactants, solvents and polymers (Monteiro et al., 2018). However, the high biodiesel production has generated a huge excess of glycerol that constitutes an environmental problem. Glycerol has limited industrial use because of impurities such as residual fats, soaps and catalysts that are expensive to remove. Furthermore, because the price of pure glycerol has dropped during the last 20 years from 1.5 to 0.50-0.60 US$/kg (Ceimici Novel Limited, 2018; Chen et al., 2018), its purification has not been encouraged. The value of crude glycerol has also oscillated from between 0 and 0.24 US$/kg (Varrone et al., 2017) which has contributed to an increased concern about the waste from the biodiesel industry. Glycerol is the most studied substrate for 1,3-PDO biological production (Varrone et al., 2017). It is formed by a reductive pathway during the fermentation (Silva et al., 2009). Because each glycerol carbon has a hydroxyl group, there is a natural tendency for reduction (Vivek et al., 2016). Several microorganisms are related to 1,3-PDO formation, such as Klebsiella pneumoniae (Drozdzyńska et al., 2014; Lee et al., 2018), Clostridium butyricum (Chatzifragkou et al., 2011; Yun et al., 2018), C. acetobutylicum (Forsberg, 1987), C. pasteurianum (Luers et al., 1997), C. beijerinckii (Wischral et al., 2016), Escherichia coli (Przystałowska et al., 2015), Citrobacter freundii and Hafnia alvei (Drozdzyńska et al., 2014), Lactobacillus brevis (Vivek et al., 2016), L. diolivorans (Pflügl et al., 2014), and L. reuteri (Ricci et al., 2015). The two most studied microorganisms are K. pneumoniae and C. butyricum (Guo et al., 2017; Sun et al., 2018). The theoretical maximum yield is 0.72 mol 1,3-PDO per mol glycerol consumed (Zeng, 1996), although higher values have been published using pure

5 cultures, which can be attributed to the co-fermentation with additional substrates and nutrients. However, the use of pure cultures has some drawbacks, such as strict operational conditions and expensive culture medium that contribute to increased production costs (Nakazawa et al., 2017); and despite the generated well-defined products compared with those of mixed cultures, these can provide a greater variety of products and may even include the shift of the major products. Therefore, to encourage the use of mixed cultures for glycerol fermentation, there is a need to define very well which parameters are important and how to control them to efficiently obtain the desired products. In the case of using mixed cultures for 1,3-PDO production, there is less data available using mixed cultures (Burniol-Figols et al., 2018; Dietz and Zeng, 2013; Gallardo et al., 2014; Jiang et al., 2017; Moscoviz et al., 2016; Varrone et al., 2017; Zhou et al., 2017). These data is not sufficient enough to ensure scaling up the process, for instance; and some operational parameters still need to be better studied in order to see their influence on maximizing the 1,3-PDO production. Therefore, there is still a need to clarify the influence of some of the operational parameters, with the aim of maximizing 1,3-PDO production. Consequently, the objective of this study is to evaluate the 1,3-PDO yield using a mixed microbial culture fixed in a silicone support in an upflow anaerobic sludge blanket (UASB) reactor, and to establish the best operational conditions using both pure and crude glycerol.

2. Material and Methods 2.1 Inoculum and medium composition The crude glycerol (purity 66 % w/w) used in this study was kindly supplied by Beta Renowable Group from their biodiesel industrial plant, located in Alicante,

6 Spain. The raw material used in that plant was recycled cooking oils. The measured pH and density of the crude glycerol samples were 10.4 and 1.088 kg.L-1, respectively. Analysis of the crude glycerol showed that almost no fat acids nor ethanol were detected, as well as no metals. Ash and salt contents of less than 1% and 0.5% were found, respectively. The remaining impurities were not identified. The original mixed culture was obtained from a lab-scale UASB reactor inoculated with granular sludge and adapted for glycerol consumption. This microbial consortium was always maintained active by means of consecutive transfers of 1 % (v/v) inoculum every four days into fresh medium in 120-mL serum bottles using a sterile hypodermic syringe. The 50-mL useful volume contained crude glycerol (15 g.L1

), macro (Varrone et al., 2013) and micronutrients (Florencio et al., 1993). One gram

NaHCO3 per g COD-glycerol was added to provide sufficient alkalinity. The transfer bottles containing the medium were closed with butyl rubber stoppers and sealed with aluminum caps; they were then purged using N2 and CO2 (80:20) for 3 min. In addition, 1 mL sodium sulfide (Na2S.9H2O, 100 g.L-1) per liter of medium was added to remove the dissolved oxygen. The bottles were autoclaved at 120 °C for 20 min prior to receiving the inoculum. The initial pH was between 7.0 and 7.5 without using reagents for pH correction. Following inoculation, the bottles were maintained under mesophilic conditions (30 ± 2 °C) and agitated at 120 rpm. 2.2 Biofilm formation Silicone hose (Carl Roth®, Germany) with a thickness, internal diameter, length, and volume of 0.15 cm and 0.40 cm, 285 cm, and 36 mL, respectively, was used for the development of attached biomass. Over a period of 4 weeks a feed solution was pumped through the tube at a flow rate of 0.3 L.d-1. Initially, the feed solution was composed of 40 g.L-1 pure glycerol (97 %, VWR Chemicals BDH Pro Lab®, Belgium), nutrients, and

7 a mixed culture (1 % v/v) at pH 7.0 to 7.5 without reagents addition. Samples were collected once a week to determine metabolite concentration. To confirm the development of biomass attached inside the hose, the procedure was repeated under similar conditions using a mineral medium with 20 g.L-1 glycerol, but without any microorganisms, which was pumped through the tube for one more week. The glycerol and metabolites were analyzed. Following this 285 cm silicone hose with the biomass attached to the inner wall was transferred and arranged in a spiral format inside the labscale UASB reactor (connected to its inlet), as shown in Fig. 1. 2.3 Reactor experimental design The system consisted of a 762-mL acrylic UASB reactor containing a 285 cm silicone hose with biofilm attached inside. The experiments were carried out for 155 days divided in two phases (P), when the reactor was fed with pure glycerol (P1) during the first 102 days, and other with crude glycerol (P2), from day 103 onwards until the end. The feed solution was prepared with the mineral medium described previously. Initially, the bioreactor was operated at influent pH of 7.5, average values for hydraulic retention time (HRT), glycerol loading rate (gly-LR), and effluent pH of 4 days, 6 ggli.L-1d-1, and 6.3, respectively. Thereafter, gly-LR was increased to values close to 22 g-gly.L-1.d-1 during P1 and 20 g-gly.L-1.d-1 for P2. During the experiments a 50 % NaOH (stock solution) was used to modify the influent pH. Influent pH changes were done during reactor operation in order to avoid effluent pH drop and to analyze possible effects on 1,3-PDO and acids production behavior. The monitoring of the reactor operational parameters i.e. flow, HRT, LR, metabolite concentration, and pH was performed daily. The reactor was placed in a temperature-controlled room at 30 ± 2 °C. 2.4 Analytical methods

8 The metabolite concentrations were determined by high performance liquid chromatography (HPLC 1200 Infinity Series, Agilent Technologies, Japan) equipped with a refractive index detector (RID) and a MetaCarb 67H column 300 x 6.5 mm (Agilent Technologies, Japan). The conditions applied to temperature, flow rate of mobile phase (H2SO4, 0.01N), and injection volume were 40 °C (both column and RID detector), 0.65 mL.min-1, and 20 µL, respectively. The product yields were calculated and analyzed in terms of mol of by-product generated per mol of glycerol consumed, as shown in Eq. (1); and volumetric production rates (productivities) for 1,3-PDO as described in Eq. (2). The average values of yield and productivity were also calculated for each operational phase for both pure and crude glycerol. In these equations, (Glyinitial - Glyfinal) is the consumption rate of glycerol. The glycerol consumption was normally described as percentage efficiency (%). For COD mass balance and products distribution, the yield (mol.mol-gly-1) of each metabolite was multiplied by the ratio of g COD.mol-1 and then, divided by the g COD.mol-1 of glycerol consumed. At the end, these values were multiplied by 100 (% COD distribution). (1)

(2)

2.5 Microbial community analyses 2.5.1 DNA extraction and Illumina sequencing Biomass samples were collected for DNA extraction and massive sequencing throughout the experiments. Samples of the inoculum (Si) and those from P1 with pure glycerol (PG) were collected on day 76 and consisted of that in the inner tube (S1PG) and that in suspension (S2PG); those from P2 with crude glycerol (CG) were collected on day 155, also from the inner tube (S1CG) and suspension (S2CG). The samples were

9 used for bacterial genomic DNA extraction using the FastDNATM SPIN Kit for soil and FastPrep® Instrument (MP Biomedicals, Santa Ana, USA). Following this, massive sequencing was carried out using the primer set 341F/806R from the total DNA samples and performed by a MySEq V3 (2X300pb) platform (Illumina, San Diego, USA) at FISABIO Sequencing and Bioinformatics Service (Valencia, Spain). 2.5.2 Phylogenetic analysis The sequence processing was performed using the Mothur package v.1.36.0 (www.mothur.org; Schloss et al., 2009). Any sequences with low quality base scores i.e. Phred quality scores < 25, were removed. Sequencing noise was removed by the Pre.cluster tool in the Mothur package and Chimeras introduced in the PCR process were detected and removed using ChimeraUquime. Qualified sequences were then clustered into operational taxonomic units (OTUs) defined by a 3 % distance level based on the distance matrix and a bootstrap higher than 60 %. Taxonomic classification was performed using the SILVA 16S rRNA gene database, using a knearest neighbor consensus and the Wang approach. Confidence values less than 80 % at a phylum level, were considered unclassified according to Wang et al. (2007). Additional statistical and graphical analyses were conducted with the package Vegan (Okasanen et al., 2010) for program R (http://www.R-project.org/). The data obtained in this study were registered with the National Center for Biotechnology Information (NCBI) under the BioProject identifier PRJNA497360. The data set containing the sequence reads was deposited in the BioSamples database, accessible under the ID numbers SRR8182192 (Si), SRR8182191 (S1PG), SRR8182190 (S2PG), SRR8182189 (S1CG), and SRR8182188 (S2CG).

3. Results and discussion

10 3.1. Biofilm formation: products yield During the fifth and last week of the biofilm formation experiment, 1,3-PDO production was observed and the yield values were between 0.44 and 0.51 (an average of 0.48) mol.mol-gly-1 with a consumption efficiency between 90 and 95 %. These values were not high when compared with the yields obtained using pure cultures, which are usually between 0.6 and 0.7 mol of 1,3-PDO.mol-gly-1 (Saxena et al., 2009; Zeng and Sabra, 2011). However, comparing them with mixed microbial culture results, the values are still above those reported in the literature, even for some of the experiments where pure cultures were used (Drozdzyńska et al., 2014; Nakazawa et al., 2017; Przystałowska et al., 2015; Zhao et al., 2006). The ratio of total acids and ethanol produced were low, at approximately 0.2 and 0.1 mol.mol-gly-1 respectively, and close to those obtained by Moscoviz et al. (2016) at a pH between 5.0 and 6.0. Acetate, followed by butyrate, were the main acids produced. The product yields during the biofilm formation were similar to those obtained using the inoculum in batch during the culture transfers using crude glycerol. In addition, the effluent pH remained between 6.0 and 6.5 without the addition of reagents. 3.2 System performance In order to evaluate the 1,3-PDO yield using an immobilized biomass in a continuous process, a UASB reactor containing silicone support medium with biofilm was used. The results obtained and the operational conditions applied during P1 and P2 phases are shown in Fig. 2 and summarized in Table 1. During the first days of P1 the product yields were close to those obtained during the biofilm formation, producing approximately 0.5 mol.mol-gly-1. Thereafter, the 1,3-PDO ratio decreased, which may have been a consequence of the initial oxygen contact during the system assembly.

11 During the first 15 days, ethanol production almost doubled from 0.1 to 0.2 mol.mol-gly-1. As the hose outlet had a small curvature (see Fig. 1), part of the biogas produced was retained inside the tube. The biogas retention may have prevented the anaerobic oxidation of ethanol into acetate (Angenent et al., 2016; Cavalcante et al., 2017), resulting in its increased production; nevertheless, acetate production was almost constant and together with butyrate they were the major volatile fatty acid (VFA) formed. After day 15, biofilm formed on external wall of the hose inside reactor was visible, an increased acetate and 1,3-PDO and a decreased ethanol and lactate production were observed. An explanation for the decrease in lactate and at least in part of ethanol is that metabolic pathways changes may have occurred. However, since this system is a new configuration, further research should be addressed on the influence of the head space or partial pressure on the products formation. The system configuration was chosen after preview experiments with the same mixed culture and substrate. In batch essays, the 1,3-PDO yield was practically constant, but not in continuous system. After 20 to 40 days of operation, the microbial activity dramatically decreased when we used different support materials like activated carbon, porous glass disks, and ceramic rings, in order to attach and retain the biomass. For each material, a range of different values of the main operational parameters was tested, but the results were also not successful. Finally, after many attempts, good results of 1,3-PDO yield and glycerol consumption efficiency, similar to those obtained in batch essays, were obtained when we used silicone hose as support material for the inside biofilm formation and development. One important observation was that the headspace formed and retained inside the hose maybe could have strongly contributed to select some microorganisms, which did not occur in the other systems and support materials tested. Therefore, the silicone hose was chosen as support material for the

12 attachment and retention of biofilm, to retain part of the biogas produced and also to prevent the washout of the microorganisms, in order to guarantee the stability and production of 1,3-PDO in continuous systems with the mixed culture studied. The change of influent pH from 7.5 to 8.5 was not favorable to the 1,3-PDO yield (Table 1). Consequently, the pH was returned again to 7.5 and gly-LR was maintained at 8 g-gly.L-1.d-1 during the days 22 to 33. As a result, an increased yield and productivity of 1,3-PDO were observed. Therefore, pH influent was maintained between 7.5 and 8.0 during most of the operation. At the end of phase P1 and beginning of P2, the pH was increased to 9, to evaluate its possible influence on the acids production using crude glycerol. However, the results showed that the reagents addition did not significantly affect the acids production, which is important in terms of process cost (discussed later). The gly-LR increased to approximately 12 g-gly.L-1.d-1 and the HRT decreased to around 2 days, an 1,3-PDO increase from 0.49 on day 33, to a maximum yield of 0.61 mol.mol-gly-1 by day 36, was observed. This maximum 1,3-PDO yield was very close to the maximum yield of 0.63 mol.mol-gly-1 obtained by Varrone et al. (2017) using mixed microbial cultures in continuous flow stirred-tank reactors (CSTR) inoculated with enriched anaerobic sludge. At the same time, the average acid yield decreased to 0.3 mol.mol-gly-1 and remained close to this value until the end of P1 on day 102. Between days 20 and 40, the yields of acetate presented higher values, coinciding with the maximum yield peak obtained for 1,3-PDO (0.61 mol.mol-gly-1). Thereafter, the yields of acetate and butyrate remained practically constant until the end of P1 and both represents the total VFA produced (~ 0.3 mol.mol-gly-1) (Fig.2). High 1,3-PDO yields are more likely to occur when acetate is the main product from oxidative pathway (Saxena et al., 2009). During that period, a high biomass inside the reactor was

13 observed. Members of the genus Clostridium were dominant and they could be responsible for the high 1,3-PDO yield (Chatzifragkou et al., 2011; Yun et al., 2018), this is discussed further in the next section. On day 79, the gly-LR change was marked by a glycerol overloading. As a consequence, the glycerol consumption dropped to approximately 80 % and the 1,3PDO yield decreased to 0.35 mol.mol-gly-1. Then, the gly-LR was reduced and maintained at approximately 22 g-gly.L-1.d-1. The 1,3-PDO yield gradually recovered and by day 103 the crude glycerol started to be used (P2). To avoid any possible inhibitory effect on the biomass due to its composition the gly-LR was halved from 22 to 11 g-gly.L-1.d-1, until day 113 (Table 1). Thereafter, the gly-LR was increased from 11 to 18 g-gly.L-1.d-1 (day 114) causing a temporary overloading, but after some time the reactor could be acclimated to the new condition. A further increase in gly-LR to approximately 20 g-gly-L-1.d-1 resulted in 1,3-PDO yield values between 0.48 and 0.50 mol.mol-gly-1. These results using either pure or crude glycerol suggest that the optimum influent pH was between 7.5 and 8 in both phases, which is very interesting for real life applications. In this study, one of the main goals was to evaluate the production of 1,3-PDO with mixed cultures using a minimum of reagents, either as nutritional medium or as for pH control, aiming to find the optimal operational conditions with lower costs. With the benefit of the process costs, the use of mixed cultures may gain more attention and practical applications for the production of 1,3-PDO from glycerol. The results obtained in this study revealed relatively optimal values for yield and productivity, either with pure or crude glycerol. Therefore, this fact shows the possibility of using crude glycerol directly and making the process even more profitable. Therefore, the best average 1,3-PDO yields using pure (0.54 mol.mol-gly-1) and

14 crude glycerol (0.48 mol.mol-gly-1) are in agreement with the data obtained using continuous system and microbial mixed consortia, but with different reactors. For example, they are higher than those found by Nakazawa et al. (2017) using pure glycerol in UASB reactor; but close to those obtained by Gallardo et al. (2014) with EGSB reactors and by Varrone et al. (2017) with CSTR reactor using crude glycerol. Some of the studies using pure cultures with or without co-fermentation, in batch or continuous assays, showed even lower yield values than those obtained in the present work, either using pure glycerol (Drozdzyńska et al., 2014; Przystałowska et al., 2015; Zhao et al., 2006) or crude glycerol (Ricci et al., 2015). Another important aspect is that both phases the experiments were conducted at a lower temperature (30 °C) and effluent pH (5.0 to 5.5), compared with that reported in other studies (37 °C and effluent pH 7.0 to 8.0). In these studies, for the maintenance of the pH range there was a need to constantly add reagent. However, this was not the case in both phases of the present experiments because an influent pH close to 7.5 representing a significant advantage in the case of large-scale 1,3-PDO production, as previously mentioned. Concerning the 1,3-PDO productivity (Table 1), the highest average values obtained using either pure (P1) or crude (P2) glycerol were approximately the same (7.4 g.L-1.d-1). The corresponding average values of gly-LR and glycerol consumption were also almost in the same range for both phases, between 18 and 20 g.L-1.d-1 and 92–93%, respectively. Productivity peaks of 8.56 and 8.13 g.L-1.d-1 were observed in P1 and P2, respectively. Therefore, due to the comparable results, it seems attractive and advantageous to use crude biodiesel-derived glycerol as substrate, in the case of large scale production of 1,3-PDO. Despite some reported restrictions due to its composition, which might inhibit some microorganisms, the use of microbial mixed consortia can overcome this drawback. As will be discussed in section 3.3, microbial population can change during the metabolic process and ensure good substrate conversion into the desired by-products.

15 Despite the good results of 1,3-PDO productivity obtained in P1 and P2, it is important to mention that the values are not so high as other data reported in the literature, especially those obtained with pure cultures using various fermentation methods. Values from 14 up to impressive 502 g.L-1.d-1 were obtained with pure cultures (Reimann et al., 1998; Saxena et al., 2009; Sun et al., 2018).When using mixed cultures, the 1,3-PDO production rates vary widely and in general they are lower than those using pure cultures. Varrone et al. (2017), using crude glycerol and anaerobic sludge in CSTR reactor obtained 5.4 and 5.2 g.L-1.d-1 by applying a one-day HRT with minimal and complete medium composition, respectively. When they applied half of that HRT, the values increased to 9.8 and 12.9 g.L-1.d-1, respectively. Gallardo et al. (2014), using EGSB reactors obtained values close to 7 g.L-1.d-1 also by applying oneday HRT. These authors mentioned values ranging from 1 to 70 g.L-1.d-1 when they used mixed cultures from granular sludge with three different forms, in natural, heattreated, and disrupted by applying different HRT values and continuous system. High production rates of 1,3-PDO were reported by Zhou et al. (2017) in batch (91 g.L-1.d-1) and fed-batch (73.4 g.L-1.d-1). Therefore, more research should be done to improve the volumetric production rate of this by-product. The reduced costs due to the reagents used (minimal nutritional medium and pH control) and biofilm use can encourage even more these new researches. The 1,3-PDO productivity is an essential parameter to indicate the proper functioning of the process mainly in continuous systems. In batch essays, metabolites and substrate concentrations cannot be neglected because they can cause inhibition to some microorganisms groups directly associated with 1,3-PDO formation. A critical glycerol concentration of 188 g.L-1 was reported for K. pneumoniae (Jiang et al., 2017); and 85 g.L-1 of glycerol and 65 g.L-1 of 1,3-PDO were cited as inhibitory concentrations

16 for C. butyricum (Colin et al., 2000). Therefore, it is advisable to avoid substrate and metabolites concentrations capable of impairing the activity of the main bacteria groups involved in the production of 1,3-PDO. On the other hand, efficient strategies for separation and purification should be chosen in the case of 1,3-PDO commercialization as biological technology from renewable resources. However, it is expected that its properties, e.g. highly hydrophilic, low volatility and high boiling point will be associated with difficulties in choosing the proper technologies, and consequently, into well-defined downstream process (Saxena et al., 2009; Sun et al., 2018). There are many separation strategies such as evaporation/distillation, electrodialysis, membranes separation, chromatography, solvent extraction, reactive extraction, salting-out extraction, and sugaring-out extraction, in which all are well described in the literature (Sun et al., 2018). These authors also highlighted the salting-out and sugaring-out extraction. According to them, the 1,3-PDO recovery was close to 98% with the salting-out extraction. Solvents recovery possibility makes this technology still more promising. The sugaring-out extraction with slightly lower 1,3-PDO recovery (82%) was attributed to be better for 1,3-PDO production from glucose. Then, to ensure the successful 1,3-PDO commercialization from biological routes, it is necessary to choose a production process and also profitable separation/ purification strategies. Fig. 3 shows the COD products distribution in terms of g COD per g COD of glycerol consumed for each gly-LR applied in phases P1 and P2. The main fermentation product in both phases was 1,3-PDO with minimal and maximal values of the total products between 40.1 and 61.3% and 42.8 and 54.9% for P1 and P2, respectively. These values are very close to those reported by Varrone et al. (2017) with anaerobic sludge and using minimal nutritional medium, and crude glycerol (60–63%). During all

17 operational phases, formate, lactate, and valerate were produced in small amounts and were included as ‘others’. Residual products such as biogas, biomass, and products not identified were represented as ‘residual’. As previously mentioned, acetate was the main short-chain VFA produced; its production seems to have increased between 14 and 18% using crude glycerol in P2 when compared with pure glycerol in P1, while butyrate decrease was observed. 3.3 Microbial community characterization After quality filtering, between 65,183 (S1PG) and 91,165 (S1CG) sequences generated by Illumina MySeq were considered for further analysis. The average lengths were 440-450 bp, which is suitable for reliable taxonomic assignment at the genera level (Table 2). Coverage, richness and evenness estimators were also calculated (Table 2). The specific richness index (Sobs) computes with the number of observed species in samples. Sobs and Chao1 revealed evidence of a higher diversity richness in the reactor fed with crude glycerol (S1CG and S2CG) when compared with either the inoculum, or that fed with pure glycerol (S1PG and S2PG). Both Shannon Indexes showed a low diversity (H<3) and a low evenness (EH<0), while the Gini Index (>0.6) in all five samples, showed evidence of a high inequality, especially in the S1PG sample. The coverage of the observed species, according to Good´s Coverage Estimator and rarefaction curves, which tend to be asymptote, showed that nearly a full census was achieved in the five samples. An overview of the bacterial taxa found in the inoculum and reactor biomass (Fig. 4 and Table 3) revealed the current low biodiversity. With regard to the level of phylum, all reads can be affiliated to only four phyla, of which Firmicutes was very dominant in all the reactor samples, between 66 and 98 %; these percentages were calculated with respect to the total reliable assigned sequences for each sample. That

18 dominance was maintained at the level of order (Clostridiales) and family (Clostridiaceae), which confirmed the lack of equitability in the reactor bacterial populations. After 100 days reactor population was already acclimated to the pure glycerol. Nearly all the bacteria attached to the inner wall of the silicone tube belonged to the family Clostridiaceae (98 %), while a greater diversity, mainly of the families Enterobacteriaceae (25 %) and Lactobacillaceae (8 %), was observed in the suspended biomass. The change from pure to crude glycerol had a drastic effect on the reactor populations. Although Clostridiaceae still formed the majority, members of the families Enterobacteriaceae (17 %) and Lactobacillaceae (9 %) were also present in the biofilm inside the silicone tube, with Lactobacillaceae becoming co-dominant in the suspended biomass. The Shannon and Gini indexes revealed that the reactor behaved as an unfair system, being the members of the former three families responsible for carrying out the conversion of glycerol to 1,3-PDO. Nevertheless, the sequences retrieved after feeding the reactor with crude glycerol showed a greater bacterial diversity than that with pure glycerol (Fig. 4, Chao index). Most of the 16S rRNA reads could reliably be assigned taxonomically at the genus level (Fig. 4b). Intra-genus analysis (Table 3) showed the evolution of the different species, depending on the substrate (pure or crude glycerol) and biomass form (attached or suspended). The focus was on Clostridium, one of the most representative and studied genus involved in the conversion of glycerol to 1,3-PDO (Chatzifragkou et al., 2011; Forsberg, 1987; Lee et al., 2015; Luers et al., 1997; Wischral et al., 2016; Yun et al., 2018). The sequences retrieved from the inoculum were assigned to C. beijerinckii (22.2 %) and C. sphenoides (8.2 %). The number of C. beijerinckii remained practically unaltered after feeding with pure glycerol (21.3% and 24.4% in S1PG and S2PG, respectively). However, C. pasteurianum became very dominant in the

19 biofilm (75.9 %), whereas it was co-dominant (22.7%) with C. beijerinckii (24.4 %) in the suspended biomass; in this, C. autoethanogenum/C. ljungdahlii were also detected (9.4%). Changing the feeding to crude glycerol had a dramatic effect on the populations of Clostridium spp., particularly on C. beijerinckii. C. pasteurianum remained as the majority species (45.4 % and 25.6 % in S1CG and S2CG, respectively), but the number of C. beijerinckii dropped dramatically to 1.6 % and 4.4 % in the attached and suspended biomass respectively. Crude glycerol derived from biodiesel has been described as toxic to C. pasteurianum and that a pre-treatment was needed for its conversion to butanol and 1,3PDO (Jensen et al., 2012). However, Jun et al. (2010) found that K. pneumoniae DSM 4799 was able to use raw glycerol as the sole carbon source for 1,3-PDO production, unlike several 1,3-PDO-producing Clostridium species that were significantly inhibited. In the present study, it is conceivable that some of the non-identified impurities in the crude glycerol may have exerted a strong inhibitory effect on C. beijerinckii and, to a lesser extent, on C. pasteurianum. Indeed, part of the Clostridium population was replaced by K. pneumoniae (17.3 %) and by different species of Lactobacillus (L. casei/L. paracasei, L. coryniformis, L. paracbuchberi) that together comprised 9.7 % of the reads in the attached biomass and by L. casei/L. paracasei (26.5 %) in the suspended biomass. Many previous works have focused on the fermentation of 1,3-PDO from glycerol using K. pneumoniae and C. butyricum (Dietz and Zeng, 2013; Guo et al., 2017; Wischral et al., 2016); however interestingly, no sequences affiliated to the latter species were retrieved in the present research. Therefore, to compare results from different works or to select a proper 1,3-PDO production system, several important factors should be considered. In the case of using pure cultures, which species are the

20 best indicated; and with mixed cultures, which biomass type (attached or suspended), glycerol form (pure or crude) and operational conditions should be selected.

4. Conclusions 1,3-PDO production with a mixed culture attached to silicone hose inside a UASB reactor is attractive with pure and crude glycerol. An average yield of 0.5 mol.mol-gly-1 with optimal effluent pH (5.0-5.5) obtained without reagent addition (influent pH 7.5-8.0), represents lower costs in practice. 1,3-PDO accounted for 61 (pure) and 55% (crude glycerol) in products distribution. A reactor exclusively fed with glycerol can be considered an extreme environment, with low biodiversity and inequitability, where Clostridium drives as the key genus. Comparing pure and crude glycerol, the last favors a greater biodiversity being Lactobacillus and Klebsiella also involved in its fermentation.

Acknowledgements This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (MINECO-FEDER/EU, CTM-2013-44734-R) and the program of Cooperación Interuniversitaria con América Latina UAM-Santander (CEAL-AL / 2017-14) to J. L. Sanz. The Brazilian agencies PDSE/CAPES (Programa de Doutorado Sanduíche, Process 88881.134642/2016-01) and Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE, Process IBPG-0194-3.07/14) provided a scholarship to S. T. S. Veras to spend a researh year in Madrid. We also wish to thank the Universidad Autónoma de Madrid (UAM, Spain) and Universidade Federal de Pernambuco (UFPE, Brazil) for their institutional support. A special acknowledgement goes to Beta Renowable Group (Elda, Spain) for kindly providing the crude glycerol used in the experiments.

21

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version.

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26 Figures captions Fig. 1 Operation flowchart for the UASB reactor coupled to a silicone tube support containing previously developed biofilm. The feeding solution was stored in a cooled reservoir at 5 to 8 °C and pumped to the reactor through a PVC tube connected directly to the silicone tube inside. The biogas produced passed through a water seal, where the volume was quantified by displacement. Fig. 2 Glycerol loading rate (Gly-LR), glycerol consumption, HRT, pH (influent and effluent), and product yields (mol.mol-gly-1) during the P1 (pure glycerol) and P2 (crude glycerol) phases. The arrows indicate the days when biomass samples were taken for molecular biology analyses. Fig. 3 COD product distribution during phases P1 and P2 in function of the applied glyLR. Formate, lactate, and valerate are included as ‘others’; and biogas, biomass, and metabolites not identified as ‘residual’. Fig. 4 Taxonomic profiles at the family (a) and genus (b) levels of the biomass used as the inoculum (Si); collected after the reactor fed with pure glycerol, attached to the inner hose (S1PG) and growing in suspension throughout the reactor (S2PG); collected after fed with crude glycerol, immobilized in the inner hose (S1CG) and growth in suspension (S2CG). Taxa with a coverage of lower than 1 % have been grouped as ‘Others’.

27

28

29

30

31 Table 1 Applied operating conditions and performance results of glycerol consumption, yield and productivity of 1,3-PDO during phases P1 (pure glycerol) and P2 (crude glycerol)

Phase P1

P2

Time (d) 1–5 6 – 21 22 – 33 34 – 53 54 – 64 65 – 76 77 – 83 84 – 102 103 – 113 114 – 124 125 – 144 145 – 155

Gly-LR (g.L-1.d-1) 5.81 ± 1.89 8.00 ± 1.72 8.00 ± 2.11 12.13 ± 0.85 14.15 ± 0.29 17.94 ± 1.10 25.02 ± 3.39 22.01 ± 0.55 11.09 ± 0.11 18.25 ± 0.52 16.35 ± 0.68 19.98 ± 0.97

HRT (d) 3.92 ± 1.69 2.78 ± 0.58 2.71 ± 0.65 1.74 ± 0.13 1.84 ± 0.11 1.57 ± 0.11 1.22 ± 0.15 1.38 ± 0.04 1.40 ± 0.02 1.45 ± 0.04 1.55 ± 0.11 1.30 ± 0.02

pH(influent)

pH(effluent)

7.5 8.5 7.5 7.6±0.1 7.5 7.5 ±0.1 7.8±0.1 8.7±0.2 9.0±0.3 8.7±0.4 8.0±0.1 8.1±0.2

6.4 ± 0.1 6.4 ± 0.4 5.1 ± 0.2 5.7 ± 0.2 5.0 ± 0.1 5.4 ± 0.1 5.0 ± 0.1 4.9 ± 0.1 5.4 ± 0.2 5.3 ± 0.1 4.9 ± 0.1 4.9 ± 0.2

Glycerol consumption (%) 98.60 ± 1.15 99.25 ± 0.73 97.89 ± 1.80 97.06 ± 2.84 93.54 ± 3.66 94.18 ± 2.23 80.69 ± 4.60 84.02 ± 4.36 93.31 ± 1.14 92.53 ± 4.87 90.64 ± 4.00 92.84 ± 4.40

Y (mol.m 0.45 0.36 0.45 0.50 0.44 0.54 0.42 0.41 0.44 0.37 0.48 0.48

32 Table 2 Coverage and estimated diversity and evenness indexes

Total reads High quality Seqs Average length Sobs Chao1 Simpson Shannon (H) Shannon even (EH) Gini Good´s coverage

Si

S1PG

S2PG

S1CG

S2CG

77,682 71,396

79,002 65,183

80,439 74,497

101,513 91,165

99,330 90,332

442 127 499 ± 265 0.24 ± 0.001 1.63 ± 0.006 0.3368

440 122 470 ± 259 0.62 ± 0.003 0.69 ± 0.007 0.1429

450 129 367 ± 164 0.19 ± 0.001 1.86 ± 0.006 0.3832

450 386 1797 ± 592 0.26 ± 0.002 1.90 ± 0.009 0.3193

452 314 1341 ± 475 0.16 ± 0.001 2.23 ± 0.007 0.3876

0.6787 99%

0.9103 99%

0.7811 99%

0.7202 99%

0.6999 99%

33 Table 3 Taxonomic assignment at specie level using the consensus sequence for each OTU. Sample OTU %1 Species Similarity2 Si

OTU1 OTU2 OTU3

22.2 8.2 0.5

Clostridium beijerinckii C. sphenoides Lactobacillus casei / L. paracasei

99 99 99

S1PG

OTU1 OTU2 OTU3

75.9 21.3 0.13

C. pasteurianum C. beijerinckii L. paracasei / L. casei

99 99 100

S2PG

OTU1 OTU2 OTU3 OTU4

24.4 22.7 9.4 8.0

C. beijerinckii C. pasteurianum C. autoethanogenum / C. ljungdahlii L. paracasei / L. casei

99 99 99 100

S1CG

OTU1 OTU2 OTU3 OTU4 OTU5 OTU6 OTU7

45.4 3.3 1.6 17.3 4.4 3.8 1.5

C. pasteurianum C. autoethanogenum / C. ljungdahlii C. beijerinckii Klebsiella pneumoniae L. paracasei / L. casei L. coryniformis L. parabuchneri

99 99 99 99 99 99 99

S2CG

OTU1 OTU2 OTU3 OTU4

25.6 5.5 4.4 26.5

C. pasteurianum C. autoethanogenum / C. ljungdahlii C. beijerinckii L. casei / L. paracasei

99 99 99 99

1 2

percentage with respect to the total sequences reliably assigned to each sample. according to BLAS resource of the NCBI database.

34 Highlights 1. UASB reactor with fixed mixed cultures proved to be highly effective and feasible. 2. 1,3-propanediol yield using pure and crude glycerol were close to 0.5 mol.mol-gly-1. 3. Crude glycerol promoted higher 1,3-propanediol productivity and microbiota diversity. 4. Clostridium pasteurianum was dominant in the biofilm attached to the support. 5. Clostridium beijerinckii is strongly inhibited by crude glycerol.