Bioconversion of crude glycerol from waste cooking oils into hydrogen by sub-tropical mixed and pure cultures

https://doi.org/10.1016/j.ijhydene.2018.02.174Get rights and content

Highlights

  • Crude Glycerol (CG) from biodiesel generation from waste cooking oils.

  • Identification of Clostridium sp. in mixed culture by Illumina MiSeq Sequencing.

  • Clostridium sp. degrades crude glycerol via oxidative pathway.

  • Enterobacter sp. degrades crude glycerol to 1,3-PD via reductive pathway.

  • Higher H2 yield during CG consumption with Clostridium sp. than Enterobacter sp.

Abstract

This study compared the biohydrogen generation by sub-tropical mixed and pure cultures from the crude glycerol from the biodiesel production using waste cooking oils (WCO). The crude glycerol was pretreated by pH adjustment. The mixed culture was obtained from a subtropical granular sludge of the UASB (Upflow Anaerobic Sludge Blanket) reactor used in the treatment of vinasse from sugarcane of ethanol and sugar industry. It was heat treated in order to inactivate hydrogen-consuming bacteria, which was identified by Illumina MiSeq Sequencing with a relative abundance of 97.96% Firmicutes Philum, 91.81% Clostridia Class and 91.81% Clostridiales Order. The pure culture was isolated from a sub-tropical granular sludge from UASB reactor of treating brewery wastewater and identified as Enterobacter sp. (KP893397). Two assays were carried in anaerobic batch reactors in order to verify the hydrogen production from crude glycerol bioconversion with: (I) mixed culture and (II) pure culture. The experiments were conducted at 37 °C, initial pH of 5.5 for assay I and 7.0 for assay II, with 20 g COD L−1 of crude glycerol. The crude glycerol consumption was 56.2% and 88.0% for the assay I and II, respectively. The hydrogen yields were 0.80 moL H2 mol−1 glycerol for the assay I and 0.13 moL H2 mol−1 glycerol for the assay II. Enterobacter sp. preferred the reductive metabolic route, generating 1460.0 mg L−1 of 1,3-propanediol, and it showed to be more sensitive in the presence of methanol from crude glycerol than mixed culture that preferred the oxidative metabolic route with biohydrogen generation. The mixed culture was more able to generate H2 than pure culture from the crude glycerol coming from the biodiesel production using WCO.

Introduction

Biodiesel is one of the biofuel sources, which can be made from waste cooking oil (WCO) in a transesterification process [1]. According to Mansir et al. (2018), about 15 million tons of waste cooking oil is annually been disposed inadequately in water or land across the globe [2]. The Basic Sanitation Company of the State of São Paulo (SABESP), in Brazil, in 2010, released a report that only 2.5–3.5% of edible vegetable oil discarded in Brazil is recycled [3]. It is estimated that each WCO liter generates 980 mL of biodiesel [4]. Despite the WCO potential for biodiesel production in Brazil, only 0.80% of this biofuel was produced from WCO in January 2017 [5].

The use of WCO represents a double benefit that makes it a very good environment-friendly feedstock. First, it is cheaper than virgin vegetable oils and second, it can be re-used and transformed, preventing their discarding into the sewer that it causes water pollution and the cost increase of water treatment [6].

According to Rodrigues et al. (2016) [7], the WCO applied as a feedstock for the biodiesel production reduces the costs for this process of 60–70%. In São Paulo, Brazil, on March/April 2017, the WCO had price of R$1.00 per liter [8], while raw virgin of soybean oil price was R$2.57 per liter [9], a price of almost 2.6 more than WCO.

The biodiesel industry has shown enormous growth in past few years [10]. This production in Brazil is encouraged by law, as established by the National Program for the Production and Use of Biodiesel, which increased with the percentage increase of biodiesel mixed with diesel oil over the years, according to Brazilian government policy [1].

Biodiesel industry generates large amounts of crude glycerol as co-product, being 10 kg for each 100 kg of biodiesel produced [11]. With global production of biodiesel crossing 20 billion liters, very large quantities of glycerol will be generated [10]. This excess of crude glycerol cannot be absorb by the traditional industries for conventional applications, such as in cosmetics, food and pharmaceutical industry, because this waste contains various impurities, such as methanol, water, soaps, free fatty acids or fatty acids methyl esters, coming from the transesterification process, and its purification is expensive, especially for small and average companies [12]. For this reason, the large quantities of crude glycerol produced each year have impacted the glycerol market, resulting in low prices of crude glycerol (1.54 US $/Kg before 2000 and 0.66 US $/Kg after 2007). Crude glycerol has been a financial and environmental liability of the biodiesel industry [7], [13].

Increasing abundance, with the biodiesel production increasing, and decreasing market price, makes crude glycerol a potential substrate for fermentation to produce hydrogen [14]. The composition in this organic matter and the basic elements that make it up is one of the reasons of why crude glycerol has been used as a promising carbon source for microbiological processes [15]. This bioconversion provides biodegradable compounds, being a benefit for the environment, promoting the improvement of the economic viability of the biodiesel industry [15], [16].

The glycerol metabolism can be given by two routes, oxidative and reductive, being known for some species such as Klebsiella sp., Citrobacter sp., Clostridium sp. and Enterobacter sp [17], [18]. In the oxidative pathway, the crude glycerol can be converted to H2 in addition to lactate, acetate, butyrate, butanol, ethanol, 2,3-butanediol and propionate, for example. In the reductive pathway, the crude glycerol is finally converted to 1,3-propanediol (1,3-PD). So, the 1,3-PD and H2 are the two major products which can be obtained by crude glycerol bioconversion [19].

Hydrogen production is attractive for its high-energy content, 141.9 MJ kg−1, and it is considered as a promising alternative to fossil fuels, producing water rather than greenhouse gases during its combustion. The 1,3-PD has important applications and it can be used as a solvent, monomers for cyclic compounds, and monomers for condensation to produce plastics [20], [21].

Most studies on fermentation process have focused on the use of pure cultures for the bioconversion of crude glycerol due to the fact that they generally exhibit higher yields than those with mixed cultures [20]. These species can be Klebsiella sp. [22], [23], Enterobacter sp. [14], [15] and Clostridium sp. [24], for example.

However, the use of pure cultures involves expensive equipment and complicated protocols. In contrast, methods with mixed cultures do not require these procedures, adopted by pure cultures, and they are easy to implement, incurring lower costs and fewer contamination problems [20].

The strains belonging to Enterobacteriaceae family and Clostridiaceae family are also potential microorganisms for bioconversion of crude glycerol into variety of products. Clostridium species have much higher potential for biohydrogen production than the Enterobacter species. The metabolism of Enterobacter species can produce maximum of 1 mol hydrogen per mole of glycerol, while metabolism of Clostridium species can produce 3 mols hydrogen per mole of glycerol [10].

The main goal of this study consisted in a comparison, has never been employed, involving the biohydrogen production between mixed and pure culture with crude glycerol from the biodiesel production in a transesterification process of used cooking oils.

Section snippets

Crude glycerol (CG)

CG was obtained from a Pilot Plant of Biodiesel Production from the Biotechnology Institute of Engineering Renewable Energy of UNIARA - University of Araraquara (Araraquara - Brazil) through transesterification of waste cooking oils with sodium hydroxide (NaOH) as catalyst and methanol as short chain alcohol for this reaction. The composition of CG (by w/w) is shown in Table 1:

The CG was pretreated by the pH adjustment to 3.0 with hydrochloric acid (1 mol L−1) to convert the soluble soap into

Mixed culture: community composition of and diversity

Illumina MiSeq Sequencing produced a total of 179255 sequences with similarity index of 97%. These sequences were assigned to 200 OTUS. Shannon index of 1.033 indicated the community diversity while Chao estimators represent the community richness of 772 [38]. Ratti et al. (2015) [39] studied the composition and diversity of the bacterial community, obtained from a granular sludge from a thermophilic UASB reactor used in the treatment of stillage from sugarcane (the same inoculum source as in

Conclusion

The Illumina MiSeq Sequencing platform allowed the characterization of the mixed culture, consisting mainly of H2-producing bacteria, such as those belonging to the Firmicutes phylum as well as those of the Clostridiales order. Thus, better results of hydrogen production with mixed culture were attributed to the predominance of Clostridiales order than the assay involving the pure culture of Enterobacter sp.

The assay containing the pure culture showed a greater utilization rate of the crude

Acknowledgments

The authors gratefully acknowledge the financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP Processes 2012/01318-01 and 2017/11767-1), CAPES and Conselho Nacional de Pesquisa e Desenvolvimento (CNPq-Proc 457144/2014-9 and 141038/2017-9) for the scholarship and CEMPEQC for support the chromatography analyses.

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