- Email: [email protected]
Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter
Bioresource Technology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter Katerina Lappa a, Panagiotis Kandylis a, Argyro Bekatorou a, Nikolaos Bastas b, Stavros Klaoudatos b, Nikolaos Athanasopoulos c, Maria Kanellaki a, Athanasios A. Koutinas a,⇑ a b c
Food Biotechnology Group, Department of Chemistry, University of Patras, 26500 Patras, Greece B.G. Spiliopoulos, 87-89 Akti Dimeon, 26333 Patras, Greece I. Athanasopoulos and Co, 106b Lontou, 26224 Patras, Greece
h i g h l i g h t s Kissiris promotes the acidogenesis of sucrose, raffinose and vinasse. Good productivity and operational stability of continuous acidogenesis of vinasse. Culture adaptation is necessary for effective vinasse acidogenesis. Butyric-type fermentation is favored during vinasse acidogenesis. The process is promising for new generation ester-based biofuels.
a r t i c l e
i n f o
Article history: Received 29 November 2014 Received in revised form 26 January 2015 Accepted 29 January 2015 Available online xxxx Keywords: Sucrose Raffinose Vinasse Acidogenesis Biofuel
a b s t r a c t The use of kissiris as promoter (culture immobilization carrier) in anaerobic acidogenesis of sucrose, raffinose and vinasse is reported. Initially, the effect of pH (4–8) and fermentation temperature (18–52 °C) on the accumulation of low molecular weight organic acids (OAs) during sucrose acidogenesis, was evaluated. The promoting effect of kissiris was confirmed compared to free cells, resulting in 80% increased OAs production. The optimum conditions (pH 8; 37 °C) were used during acidogenesis of sucrose/raffinose mixtures. A continuous system was also operated for more than 2 months. When sucrose and sucrose/raffinose mixtures were used, lactic acid type fermentation prevailed, while when vinasse was used, butyric acid type fermentation occurred. Total OAs concentrations were more than 14 g/L and ethanol concentrations were 0.5–1 mL/L. Culture adaptation in vinasse was necessary to avoid poor results. The proposed process is promising for new generation ester-based biofuel production from industrial wastes. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Vinasse is a high organic load waste of ethanol producing industries, which use molasses as substrate (9–14 L of vinasse produced per liter of ethanol) (Goldemberg et al., 2008; España-Gamboa et al., 2012). It is produced in large amounts, since 29% of the total ethanol produced in European Union comes from the use of molasses (Balat and Balat, 2009). The treatment of vinasse prior to its disposal is a necessity and various methods such as fertirrigation have been proposed although they suffer from several limitations (Wheals et al., 1999; Navarro et al., 2000). Exploitation of ⇑ Corresponding author. Tel.: +30 2610997104; fax: +30 2610997105. E-mail address: [email protected] (A.A. Koutinas).
vinasse in a biorefinery manner may include its use as fermentation substrate for the production of added value co-products, such as low molecular weight organic acids (OAs) and ethanol for application in novel ester-based biofuel production. This approach could reduce the problem of vinasse disposal and enhance the sustainability of sugarcane-to-ethanol plants. OAs such as acetic, propionic, iso-bytyric, butyric, iso-valeric and valeric acid, have many applications, including biogas and biodiesel production (Fontanille et al., 2012), and are mainly produced through chemical synthesis using fossil fuels (Eggeman and Verser, 2005). Recently their production through anaerobic fermentation of waste biomass has been proposed. This alternative has the advantage of being a cost-effective and environmentally friendly process. Volatile fatty acids (VFAs) are produced mainly during
http://dx.doi.org/10.1016/j.biortech.2015.01.131 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131
2
K. Lappa et al. / Bioresource Technology xxx (2015) xxx–xxx
the second step of anaerobic digestion, the acidogenesis step (De la Rubia et al., 2009), however, the production of lactic, succinic and other OAs has also been reported. Other fermentation products during acidogenesis are alcohols such as methanol and ethanol, carbonyl compounds, carbon dioxide and hydrogen (Grimmler et al., 2011; Li et al., 2013). Contino et al. (2011) evaluated the use of ethyl esters of low molecular weight VFAs in a homogenous charge compression ignition engine with promising results. Process parameters such as pH, temperature, the C/N ratio and hydraulic retention time play an important role, controlling the production of OAs during fermentation (Chen et al., 2013). In previous studies it was also shown that methane, alcoholic and acidogenic fermentations can be promoted in the presence of porous materials such as c-alumina pellets and kissiris (a porous volcanic rock found in Greece; 70% SiO2) acting as culture immobilization carriers (Koutinas et al., 1991; Tsoutsas et al., 1990; Galanakis et al., 2012; Syngiridis et al., 2013, 2014). The results showed that under selected process conditions (substrate concentration and pH, temperature, type of substrate) the anaerobic acidogenesis can lead to products with different compositions of OAs and ethanol, giving the possibility to produce different types of ester-based new generation biofuels (Syngiridis et al., 2013, 2014). Based on the above studies, the aim of the present study was to examine the effect of kissiris as immobilization carrier and promoter on the anaerobic acidogenesis of sucrose and sucrose/raffinose mixtures, to study the operational stability of the continuous acidogenesis of these sugars, and subsequently of vinasse, and to determine the OAs chemical composition and degree of ethanol formation.
2.4. Cell immobilization and continuous anaerobic acidogenic fermentation of sucrose The experimental apparatus used for cell immobilization and for continuous acidogenic fermentation consisted of a 1.25 L glass tower reactor placed in an incubator set at 37 °C, and connected to a high accuracy peristaltic pump. The bioreactor was filled with 950 g mineral kissiris and equal volumes of 20 g/L sucrose medium and anaerobic culture suspension. The system was left to ferment for two days at 37 °C without feeding in order to achieve cell immobilization. Subsequently, fresh sucrose medium was pumped continuously into the bioreactor and a steady state was obtained in a few days. The bioreactor was pumped continuously and successively with sucrose, sucrose and raffinose mixture and vinasse media as described below. Samples were removed at various time intervals and analyzed for OAs, ethanol and residual sugar by GC and HPLC. 2.5. Media for continuous acidogenic fermentation The various substrates that were used successively for the continuous anaerobic fermentation were: (1) 20 g/L sucrose medium for 17 days, (2) 17 g/L sucrose–3 g/L raffinose medium for 16 days, (3) 17 g/L sucrose–3 g/L raffinose–1:5 vinasse:water dilution for 8 days, (4) 1:5 vinasse:water dilution for 9 days, and finally (5) 1:10 vinasse:water dilution for 10 days. All media contained aqueous NH3 and 50% H3PO4 (COD:N:P ratio of 100:5:1), 4 g/L NaHCO3 and 4 g/L yeast extract and had pH 8. They were sterilized by autoclaving at 120 °C for 10 min. For the vinasse media only pH adjustment took place and there was no sterilization.
2. Methods
2.6. Analysis of ethanol
2.1. Culture and growth media
Ethanol was determined on a Shimadzu GC-8A system, with a Teknokroma HAYE SEP Q 80/100 column, a C-R6A Chromatopack integrator, He as carrier gas (40 mL/min), and a FID detector. The injection port and detector temperature was 210 °C. The column temperature was 130 °C. Samples of 2 mL were injected directly into the column. Determinations were done by means of standard curves.
Mixed bacterial anaerobic culture obtained from a UASB reactor (Syngiridis et al., 2013, 2014) was inoculated in a medium containing 50 g/L glucose, aqueous NH3 and 50% H3PO4 (COD:N:P ratio of 100:5:1), 4 g/L NaHCO3, 4 g/L yeast extract, and without pH adjustment (Koutinas et al., 1991). Cell growth was carried out at 37 °C. The medium was sterilized by autoclaving at 120 °C for 10 min.
2.7. Analysis of OAs 2.2. Batch acidogenic fermentation of sucrose at different pH values For the repeated batch fermentations conical flasks were used containing immobilized mixed anaerobic culture on 100 g mineral kissiris and 150 mL synthetic medium containing 40 g/L sucrose, aqueous NH3 and 50% H3PO4 (COD:N:P ratio of 100:5:1), 4 g/L NaHCO3, 4 g/L yeast extract. Fermentations were carried out at 37 °C and at different pH values from 4 to 8. The media were sterilized by autoclaving at 120 °C for 10 min. 2.3. Batch acidogenic fermentation of sucrose and raffinose mixtures For the repeated batch fermentations conical flasks were used containing immobilized mixed anaerobic culture on 100 g mineral kissiris and 100 mL synthetic medium containing 20 g/L sugars. Fermentations were carried out at 37 °C with initial pH 8. Synthetic media with five different compositions of sugars were used: (1) 20 g/L sucrose, (2) 14 g/L sucrose and 6 g/L raffinose, (3) 10 g/L sucrose and 10 g/L raffinose, (4) 6 g/L sucrose and 14 g/L raffinose, and (5) 20 g/L raffinose. Each medium also contained aqueous NH3 and 50% H3PO4 (COD:N:P ratio of 100:5:1), 4 g/L NaHCO3, and 4 g/L yeast extract. All media were sterilized by autoclaving at 120 °C for 10 min.
OAs were determined by High Performance Liquid Chromatography (HPLC), using a Jasco LC-2000 Plus chromatograph (Jasco Inc., Japan) with a Bio-rad Aminex HPX-87H column (300 7.8 mm i.d., 9 lm particle size), a PU-2089 plus quaternary gradient pump, a CO-2060 Plus oven set at 50 °C, a MD-2018 plus photodiode array detector operated at 210 nm and an AS 2050 PLUS autosampler. A solution of 0.008 N H2SO4 was used as mobile phase with a flow rate of 0.6 mL/min. The samples were filtered through disposable syringe cellulose acetate filters (CHROMAFIL) with 0.20 nm pore size and all the data were processed with ChromNav program. All determinations were done by means of standard curves. 2.8. Determination of residual sugar Residual sugar was determined by HPLC on a Shimadzu chromatograph with a NUCLEOGEL ION 300 OA column, a LC-9A pump, a CTO-10A oven at 30 °C and a RID-6A refractive index detector. H2SO4 0.008 N was used as mobile phase with a flow rate of 0.8 mL/min and propanol-1 was used as an internal standard. A volume of 0.25 mL of sample and 0.625 mL of 1% (v/v) solution of propanol-1 were diluted to 25 mL. The samples were filtered with a disposable syringe cellulose acetate filter (CHROMAFIL) with 0.20 nm pore size and then 60 lL of the final solution were injected
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131
3
0.48 ± 0.16 0.43 ± 0.07 0.42 ± 0.03 0.42 ± 0.08 0.37 ± 0.09 9.57 ± 1.11 8.34 ± 1.20 7.82 ± 0.59 7.33 ± 1.36 6.53 ± 1.57 – – – – – – 0.17 ± 0.10 – – – 1.14 ± 0.41 1.03 ± 0.25 0.92 ± 0.15 0.96 ± 0.10 1.51 ± 0.09 7.93 ± 3.56 6.39 ± 0.86 6.42 ± 0.58 6.08 ± 1.29 4.49 ± 1.46 0.11 ± 0.10 0.24 ± 0.20 0.10 ± 0.10 – 0.08 ± 0.08 – – – – – 0.39 ± 0.16 0.68 ± 0.34 0.39 ± 0.16 0.29 ± 0.09 0.45 ± 0.12 – 0.80 ± 0.26 1.20 ± 0.53 2.50 ± 0.63 2.50 ± 0.78
5.33 ± 1.00 5.67 ± 0.51 4.30 ± 0.20 5.40 ± 0.44 4.20 ± 0.53
3.23 ± 0.46 3.27 ± 0.25 3.21 ± 0.18 3.16 ± 0.02 3.52 ± 0.24 Without kissiris 100 suc 70 suc/30 raf 50 suc/50 raf 30 suc/70 raf 100 raf
6.26 ± 5.13 1.50 ± 1.06 1.00 ± 078 0.40 ± 0.17 0.37 ± 0.06
9.93 ± 1.57 10.60 ± 2.05 7.65 ± 1.69 12.44 ± 0.93 10.75 ± 0.67 4.99 ± 3.70 5.81 ± 1.94 2.86 ± 2.60 6.75 ± 0.69 5.07 ± 1.98 0.12 ± 0.10 0.23 ± 0.20 – – 0.11 ± 0.11 0.89 ± 0.06 0.94 ± 0.09 1.03 ± 0.09 0.98 ± 0.14 1.00 ± 0.09 4.48 ± 4.30 0.71 ± 0.43 3.43 ± 0.61 1.14 ± 1.02 3.49 ± 1.78 0.28 ± 0.20 0.78 ± 0.29 0.43 ± 0.10 0.50 ± 0.07 0.27 ± 0.23 0.18 ± 0.16 1.17 ± 0.92 0.20 ± 0.19 1.58 ± 0.97 0.26 ± 0.25
Propionic
With kissiris 100 suc 70 suc/30 raf 50 suc/50 raf 30 suc/70 raf 100 raf
Acetic
OAs (g/L) Residual sugar (g/L) Ethanol (mL/L) Final pH
Batch acidogenesis was performed at 37 °C and at pH 8 as the optimum conditions. Culture immobilization on kissiris increased
Substrate composition (%)
3.2. Effect of immobilization on OAs and ethanol production during acidogenesis of sucrose and raffinose mixtures
Table 1 Batch acidogenesis of sucrose (suc) and raffinose (raf) mixtures using anaerobic mixed culture immobilized on kissiris.
Since it is well established that factors such as pH and fermentation temperature control affect the production of OAs during anaerobic fermentation (Chen et al., 2013), an optimization study of these parameters took place using cells immobilized on kissiris and free cells and sucrose as substrate. In order to identify the effect of pH (4–8), repeated batch acidogenic fermentations of sucrose (40 g/L) were performed at 37 °C. At all pH values the use of kissiris led to increased OAs concentration. Specifically, the production of OAs increased with increasing pH, with the highest concentration (23 g/L) obtained at pH 8, which is in accordance with previous studies (Jiang et al., 2013; Wang et al., 2014). The main acid produced using both free and immobilized cells was lactic acid (more than 50% of total OAs) followed by succinic acid and acetic acid. A small amount of ethanol was also produced varying from 1.6 (at pH 8) to 6.5 mL/L (at pH 6) for free cells and from 1.7 (at pH 8) to 3.4 mL/L (at pH 6) for cells immobilized on kissiris. These results suggest that ethanol-type fermentation also took place. In order to evaluate the effect of temperature, fermentations were also carried out at 18–52 °C, with substrate of pH 7 for free cells and pH 8 for immobilized cells. In all cases the promotional effect of kissiris was observed leading to almost two-fold higher OAs concentrations. In the case of kissiris, an increase in OAs concentrations was observed with the increase of temperature from 18 °C to 37 °C. The OAs concentration at 52 °C (10.1 g/L) was lower than that at 37 °C (14.9 g/L), indicating a lower acidogenic activity at higher temperatures, which is in accordance to other studies (Jiang et al., 2013; Komemoto et al., 2009). The main acid was lactic acid (more than 50% of total OAs) indicating that lactate-type fermentation occurred as was also observed in other studies (Wang et al., 2014).
Butyric
3.1. Effect of pH and fermentation temperature on OAs and ethanol production during sucrose acidogenesis
0.27 ± 0.08 1.21 ± 0.59 0.66 ± 0.25 1.50 ± 0.84 0.55 ± 0.06
Lactic
Succinic
Isovaleric
Valeric
The last few years, research has focused on the production of new generation biofuels based on ester production using the simultaneously produced OAs and ethanol during acidogenic fermentation of carbohydrates (Ren et al., 1997). Furthermore, in recent studies the promotional effect of porous materials serving as culture immobilization carriers, such as c-alumina, on glucose acidogenesis was also shown (Syngiridis et al., 2013, 2014). These works open the way for the production of the new generation biofuels from agro-industrial wastes. Vinasse the liquid waste of ethanol distilleries, contains sucrose and raffinose as residual sugar. Therefore, as a first step to evaluate vinasse as substrate for esters production, the study of sucrose and raffinose acidogenesis was necessary. Mineral kissiris, a volcanic rock found in Greece, is a very cheap and abundant porous material compared to c-alumina. Therefore the objectives of this study were to investigate any promotional effects of kissiris, as compared with free cells, on the acidogenesis of sucrose and raffinose mixtures and vinasse regarding the composition of the accumulating OAs and ethanol formation in batch and continuous operation.
– – – – –
Total (g/L)
3. Results and discussion
0.17 ± 0.12 0.17 ± 0.06 0.30 ± 0.17 0.27 ± 0.06 0.20 ± 0.15
Yield (g/g)
directly to the column. Residual sugar concentrations were calculated using standard curves.
0.50 ± 0.18 0.53 ± 0.13 0.38 ± 0.12 0.62 ± 0.05 0.54 ± 0.03
K. Lappa et al. / Bioresource Technology xxx (2015) xxx–xxx
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131
4
K. Lappa et al. / Bioresource Technology xxx (2015) xxx–xxx
Fig. 1. OAs composition of the effluents produced during the continuous acidogenesis of various influents, at 37 °C and initial pH 8, using mixed anaerobic culture immobilized on kissiris.
Table 2 Continuous acidogenesis of sucrose (suc) and sucrose/raffinose (raf) media using anaerobic mixed culture immobilized on kissiris. Days (d)
Final pH
Ethanol (ml/L)
OAs (g/L) Acetic
Propionic
Isobutyric
Butyric
Lactic
Succinic
Isovaleric
Total
Yield (g/g)
Productivity (g/Ld)
0.90 0.89 0.30 0.18 1.05 3.02 0.81 0.34 0.49 0.39 0.55 0.06 0.03 1.66
0.31 0.29 0.24 0.41 0.38 0.24 0.20 0.54 0.32 0.29 0.23 0.19 – 0.21
– – – – – – – – – – – – – –
– – – – – – – – – – – – – –
– – 0.35 0.48 0.80 1.06 0.96 3.34 1.01 0.69 0.39 0.27 – –
9.18 9.16 9.68 7.01 9.88 7.58 5.21 6.58 7.00 7.94 8.90 6.74 7.98 8.34
– – – – 0.88 0.94 0.93 0.89 0.96 1.07 1.21 1.22 1.67 1.45
– – – – – – – – – – – – – 0.28
9.49 9.45 10.27 7.90 11.94 9.82 7.30 11.35 9.29 9.99 10.73 8.42 9.65 11.58
0.47 0.47 0.51 0.40 0.60 0.49 0.37 0.57 0.47 0.50 0.54 0.42 0.48 0.60
3.80 3.78 4.11 3.16 4.78 3.93 2.92 4.54 3.72 4.00 4.29 3.37 3.86 4.63
Feed: suc 17 g/L/raf 3 g/L 20 5.0 0.85 21 4.3 0.42 22 4.4 1.32 23 4.6 0.10 27 4.7 1.27 28 4.3 0.50 31 3.7 2.75 32 4.0 0.30 33 4.1 2.60
0.46 0.40 0.38 0.28 0.45 0.29 0.19 – –
0.32 0.22 – – – – – – –
1.38 1.34 1.35 1.22 1.56 1.24 – – –
– – – – – – – – –
4.03 6.26 6.49 5.77 4.98 5.61 5.26 5.48 7.57
1.19 0.91 – – 0.87 0.90 0.90 0.99 0.93
– – 0.47 0.53 – – – – –
7.38 9.13 8.69 7.80 7.86 8.04 6.35 6.47 8.50
0.37 0.46 0.43 0.39 0.39 0.40 0.32 0.32 0.43
2.95 3.65 3.48 3.12 3.14 3.22 2.54 2.59 3.40
Feed: suc 20 g/L 1 3.8 2 4.2 3 4.6 4 5.3 5 4.6 7 4.4 8 4.6 9 4.7 10 4.4 12 4.4 13 4.2 14 4.9 16 4.5 17 4.3
the OAs yield in most of the tested sucrose/raffinose mixtures as compared with free cells. The highest improvement was observed in the case of 100% raffinose and the 30% sucrose–70% raffinose mixture. Table 1 shows the results of the batch acidogenesis of sucrose and raffinose mixtures with and without the use of kissiris. Bioconversion of sucrose and raffinose occurred in all fermentation batches indicating the capability of the mixed anaerobic culture to ferment this type of sugars. The OAs yield factor varied from 0.38 to 0.62 g/g for an initial sugar concentration of 20 g/L. OAs concentration ranged from 5.9 to 13.4 g/L, while ethanol formation did not exceed 0.5 mL/L. Without kissiris the OAs concentrations and yield factors were lower, while higher concentrations of ethanol were observed. OAs that were formed were lactic, valeric, acetic, propi-
onic, succinic, and butyric acids, and in few cases isovaleric acid. Lactic acid and valeric acid were the predominant acids in most cases. These results encouraged the subsequent study of continuous acidogenesis using immobilized cells in order to achieve the aforementioned objectives. 3.3. OAs and ethanol production during continuous acidogenesis of sucrose and sucrose/raffinose mixtures Continuous acidogenesis was performed using successively sucrose, sucrose/raffinose mixtures and finally vinasse solutions (Fig. 1). During the continuous fermentation of sucrose and sucrose/raffinose mixtures (Table 2), lactic acid was produced at
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131
5
K. Lappa et al. / Bioresource Technology xxx (2015) xxx–xxx Table 3 Continuous acidogenesis of vinasse using anaerobic mixed culture immobilized on kissiris. Days (d)
Final pH
Ethanol (ml/L)
OAs (g/L) Acetic
Propionic
Isobutyric
Butyric
Lactic
Succinic
Isovaleric
1.21 1.37
– –
– –
– –
– –
4.50 5.45
– 0.81
– –
4.50 6.26
1.80 2.50
Feed: 50% vinasse 39 4.5
2.92
–
–
–
–
3.52
0.84
–
4.36
1.74
Feed: 75% vinasse 41 5.0
1.27
0.37
–
–
–
7.58
–
–
7.95
3.18
Feed: 100% vinasse (1:5) 43 5.8 44 6.6 46 6.6 47 6.7 48 6.3 49 6.2 50 6.9
1.10 – 0.71 0.80 3.12 1.04 1.32
– 3.36 3.48 3.68 2.91 3.19 2.60
– 1.30 – 0.97 – – –
– 1.11 1.63 1.52 1.52 1.51 1.34
3.19 7.56 7.74 8.65 7.45 8.52 7.79
2.44 – – – – – –
– – – – – – –
– – – 0.51 – – –
5.64 13.33 12.85 15.33 11.88 13.22 11.73
2.26 5.33 5.14 6.13 4.75 5.29 4.69
Feed: 100% vinasse (1:10) 53 6.8 1.35 54 7.9 0.10 55 7.9 2.07 57 7.8 0.34 60 6.7 0.30
1.12 0.99 1.59 0.87 1.66
– – – – –
– – – – –
2.40 3.10 4.02 4.13 3.07
– – – – –
– – – – –
– – – – –
3.52 4.09 5.61 5.00 4.73
1.41 1.64 2.24 2.00 1.89
Feed: 25% vinasse 35 4.1 37 3.9
higher amounts compared with the other acids formed (acetic, propionic, isobutyric, butyric, succinic and isovaleric). The total OAs concentration varied between 6.4 and 12 g/L. Lactic acid accounted for about 80% of total OAs while butyric acid production was very low and occurred only during the first 14 days of operation. There was no butyric acid production observed when sucrose–raffinose mixtures were used. Ethanol production was low (up to 3 mL/L). The sugar conversion was 100%. 3.4. OAs production during continuous acidogenesis of vinasse After more than a month of operation, vinasse was used as influent in the continuous system and the results are summarized in Table 3. Firstly a dilution of concentrated vinasse 1:5 with water was used for 9 days simulating the content of fresh vinasse. The use of vinasse caused an increase on the total OAs content, which reached 15.3 g/L and also changed their composition (Table 3 and Fig. 1). The lactic acid concentration decreased instantly and subsequently butyric acid and acetic acid concentration increased to 7.3 and 2.7 g/L, respectively. Butyric acid accounted for 60% of total OAs and acetic acid for 23%. Ethanol production was not affected and concentrations up to 3.1 mL/L were reported. The operation was continued for ten more days using a 1:10 dilution ratio of concentrated vinasse with water (accounting for a 1:2 dilution of fresh vinasse). This led to a decrease in total OAs to a mean value of 4.6 g/L, while only butyric and acetic acids were produced. 3.5. Discussion Mineral kissiris is a volcanic siliceous and porous material of very low cost. In various previous studies it has been indicated as a suitable material for use as cell immobilization carrier and promoter of alcoholic fermentation processes (Kourkoutas et al., 2004). Therefore, the idea was to further examine this material as promoter of the acidogenesis of vinasse. The effect of pH and temperature conditions were initially studied, showing that the best pH was 8 and the optimum temperature 37 °C, leading to an 80% improvement of yield when kissiris was used. These results encouraged the study of continuous acidogenesis of vinasse (Tables 2 and 3). However, due to the poor results obtained when vinasse
Total
Productivity (g/Ld)
was used directly, adaptation of the culture was done using successively sucrose, sucrose/raffinose mixtures, sucrose/raffinose/vinasse mixtures and finally vinasse. This adaptation process led to a substantial improvement of the acidogenesis of vinasse with an operational stability of at least two months. Table 3 shows that the predominant acid was butyric acid and the ethanol which was formed simultaneously had an average concentration of 1 mL/L, suggesting that it could esterify about the 15% of the produced OAs. In order to cover the amount of ethanol required for esterification of the total amount of OAs, the study of a system consisting of an additional bioreactor containing immobilized Saccharomyces cerevisiae and fed with carbohydrate containing wastes is proposed. The treatment of vinasse led to a 15 g/L OAs concentration, which could give about 25 mL/L esters. Suggesting that the esterification would be performed in a non-polar organic solvent using lipases, the production of an ester-based biofuel could be done with low energy demand. Because esters are non-polar and can form a second layer on the aqueous phase, their separation could also be done with low energy consumption when they are produced by chemical esterification. 4. Conclusions The use of kissiris as mixed anaerobic culture immobilization carrier promoted the batch and continuous acidogenic fermentation of sucrose and raffinose mixtures. The system presented high operational stability, and the successful adaptation of the biocatalyst in sucrose/raffinose media resulted to the possibility for scale-up of the acidogenesis of vinasse. Lactic acid-type fermentation occurred in synthetic sugar media, while butyric acid-type fermentation occurred when vinasse was used. The acidogenesis of vinasse in the presence of kissiris and after an adaptation treatment is promising for a new generation ester-based biofuel technology development. Acknowledgements Research project co-financed by the European Union (European Regional Development Fund – ERDF) and Greek National Funds through the Operational Program ‘‘Competitiveness and
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131
6
K. Lappa et al. / Bioresource Technology xxx (2015) xxx–xxx
Entrepreneurship’’ of the National Strategic Reference Framework (NSRF) 2007–2013 – National Action ‘‘Cooperation 2011: Partnerships of Production and Research Institutions in Focused Research and Technology Sectors’’ of General Secretariat for Research and Technology (Contract Nr. 11SYN-8-839). References Balat, M., Balat, H., 2009. Recent trends in global production and utilization of bioethanol fuel. Appl. Energy 86, 2273–2282. Chen, H., Meng, H.J., Nie, Z.C., Zhang, M.M., 2013. Polyhydroxyalkanoate production from fermented volatile fatty acids: effect of pH and feeding regimes. Bioresour. Technol. 128, 533–538. Contino, F., Foucher, F., Mounaim-Rousselle, C., Jeanmart, H., 2011. Combustion characteristics of tricomponent fuel blends of ethyl acetate, ethyl propionate, and ethyl butyrate in homogeneous charge compression ignition (HCCI). Energy Fuel 25, 1497–1503. De La Rubia, M.A., Raposo, F., Rincón, B., Borja, R., 2009. Evaluation of the hydrolytic–acidogenic step of a two-stage mesophilic anaerobic digestion process of sunflower oil cake. Bioresour. Technol. 100, 4133–4138. Eggeman, T., Verser, D., 2005. Recovery of organic acids from fermentation broths. Appl. Biochem. Biotechnol. 121–124, 605–618. España-Gamboa, E.I., Mijangos-Cortés, J.O., Hernández-Zárate, G., Maldonado, J.A.D., Alzate-Gaviria, L.M., 2012. Methane production by treating vinasses from hydrous ethanol using a modified UASB reactor. Biotechnol. Biofuels 5, 82. Fontanille, P., Kumar, V., Christophe, G., Nouaille, R., Larroche, C., 2012. Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Yarrowia lipolytica. Bioresour. Technol. 114, 443–449. Galanakis, C.M., Kordulis, C., Kanellaki, M., Koutinas, A.A., Bekatorou, A., Lycourghiotis, A., 2012. Effect of pressure and temperature on alcoholic fermentation by Saccharomyces cerevisiae immobilized on c-alumina pellets. Bioresour. Technol. 114, 492–498. Goldemberg, J., Coelho, S.T., Guardabassi, P., 2008. The sustainability of ethanol production from sugarcane. Energy Policy 36, 2086–2097. Grimmler, G., Janssen, H., Krauße, D., Fischer, R.-J., Bahl, H., Dürre, P., Liebl, W., Ehrenreich, A., 2011. Genome-wide gene expression analysis of the switch
between acidogenesis and solventogenesis in continuous cultures of Clostridium acetobutylicum. J. Mol. Microbiol. Biotechnol. 20, 1–15. Jiang, J., Zhang, Y., Li, K., Wang, Q., Gong, C., Li, M., 2013. Volatile fatty acids production from food wastes: Effects of pH, temperature, and organic loading rate. Bioresour. Technol. 143, 525–530. Komemoto, K., Lim, Y.G., Nagao, N., Onoue, Y., Niwa, C., Toda, T., 2009. Effect of temperature on VFAs and biogas production in anaerobic solubilization of food waste. Waste Manag. 29, 2950–2955. Kourkoutas, Y., Bekatorou, A., Banat, I.M., Marchant, R., Koutinas, A.A., 2004. Immobilization technologies and support materials suitable in alcohol beverages production: a review. Food Microbiol. 2, 377–397. Koutinas, A.A., Toutoutzidakis, G., Kana, K., Kouinis, I., 1991. Methane fermentation promoted by gamma-alumina pellets. J. Ferment. Bioeng. 72, 64–67. Li, Y., Hua, D., Zhang, J., Zhao, Y., Xu, H., Liang, X., Zhanq, X., 2013. Volatile fatty acids distribution during acidogenesis of algal residues with pH control. World J. Microb. Biotechnol. 29, 1067–1073. Navarro, A.R., Sepúlveda, M.del C., Rubio, M.C., 2000. Bio-concentration of vinasse from the alcoholic fermentation of sugar cane molasses. Waste Manag. 20, 581– 585. Ren, N.Q., Wang, B.Z., Huang, J.C., 1997. Ethanol-type fermentation from carbohydrate in high rate acidogenic reactor. Biotechnol. Bioeng. 54, 428–433. Syngiridis, K., Bekatorou, A., Kallis, M., Kandylis, P., Kanellaki, M., Koutinas, A.A., 2013. c-Alumina as a process advancing tool for a new generation biofuel. Bioresour. Technol. 132, 45–48. Syngiridis, K., Bekatorou, A., Kandylis, P., Larroche, C., Kanellaki, M., Koutinas, A.A., 2014. Favouring butyrate production for a new generation biofuel by acidogenic glucose fermentation using cells immobilised on c-alumina. Bioresour. Technol. 161, 118–123. Tsoutsas, T., Kanellaki, M., Psarianos, C., Kalliafas, A., Koutinas, A.A., 1990. Kissiris-a mineral support for the promotion of ethanol fermentation by Saccharomycescerevisiae. J. Ferment. Bioeng. 69, 93–97. Wang, K., Yin, J., Shen, D., Li, N., 2014. Anaerobic digestion of food waste for volatile fatty acids (VFAs) production with different types of inoculum: Effect of pH. Bioresour. Technol. 161, 395–401. Wheals, A.E., Basso, L.C., Alves, D.M.G., Amorim, H.V., 1999. Fuel ethanol after 25 years. Trends Biotechnol. 17, 482–487.
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter Katerina Lappa a, Panagiotis Kandylis a, Argyro Bekatorou a, Nikolaos Bastas b, Stavros Klaoudatos b, Nikolaos Athanasopoulos c, Maria Kanellaki a, Athanasios A. Koutinas a,⇑ a b c
Food Biotechnology Group, Department of Chemistry, University of Patras, 26500 Patras, Greece B.G. Spiliopoulos, 87-89 Akti Dimeon, 26333 Patras, Greece I. Athanasopoulos and Co, 106b Lontou, 26224 Patras, Greece
h i g h l i g h t s Kissiris promotes the acidogenesis of sucrose, raffinose and vinasse. Good productivity and operational stability of continuous acidogenesis of vinasse. Culture adaptation is necessary for effective vinasse acidogenesis. Butyric-type fermentation is favored during vinasse acidogenesis. The process is promising for new generation ester-based biofuels.
a r t i c l e
i n f o
Article history: Received 29 November 2014 Received in revised form 26 January 2015 Accepted 29 January 2015 Available online xxxx Keywords: Sucrose Raffinose Vinasse Acidogenesis Biofuel
a b s t r a c t The use of kissiris as promoter (culture immobilization carrier) in anaerobic acidogenesis of sucrose, raffinose and vinasse is reported. Initially, the effect of pH (4–8) and fermentation temperature (18–52 °C) on the accumulation of low molecular weight organic acids (OAs) during sucrose acidogenesis, was evaluated. The promoting effect of kissiris was confirmed compared to free cells, resulting in 80% increased OAs production. The optimum conditions (pH 8; 37 °C) were used during acidogenesis of sucrose/raffinose mixtures. A continuous system was also operated for more than 2 months. When sucrose and sucrose/raffinose mixtures were used, lactic acid type fermentation prevailed, while when vinasse was used, butyric acid type fermentation occurred. Total OAs concentrations were more than 14 g/L and ethanol concentrations were 0.5–1 mL/L. Culture adaptation in vinasse was necessary to avoid poor results. The proposed process is promising for new generation ester-based biofuel production from industrial wastes. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Vinasse is a high organic load waste of ethanol producing industries, which use molasses as substrate (9–14 L of vinasse produced per liter of ethanol) (Goldemberg et al., 2008; España-Gamboa et al., 2012). It is produced in large amounts, since 29% of the total ethanol produced in European Union comes from the use of molasses (Balat and Balat, 2009). The treatment of vinasse prior to its disposal is a necessity and various methods such as fertirrigation have been proposed although they suffer from several limitations (Wheals et al., 1999; Navarro et al., 2000). Exploitation of ⇑ Corresponding author. Tel.: +30 2610997104; fax: +30 2610997105. E-mail address: [email protected] (A.A. Koutinas).
vinasse in a biorefinery manner may include its use as fermentation substrate for the production of added value co-products, such as low molecular weight organic acids (OAs) and ethanol for application in novel ester-based biofuel production. This approach could reduce the problem of vinasse disposal and enhance the sustainability of sugarcane-to-ethanol plants. OAs such as acetic, propionic, iso-bytyric, butyric, iso-valeric and valeric acid, have many applications, including biogas and biodiesel production (Fontanille et al., 2012), and are mainly produced through chemical synthesis using fossil fuels (Eggeman and Verser, 2005). Recently their production through anaerobic fermentation of waste biomass has been proposed. This alternative has the advantage of being a cost-effective and environmentally friendly process. Volatile fatty acids (VFAs) are produced mainly during
http://dx.doi.org/10.1016/j.biortech.2015.01.131 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131
2
K. Lappa et al. / Bioresource Technology xxx (2015) xxx–xxx
the second step of anaerobic digestion, the acidogenesis step (De la Rubia et al., 2009), however, the production of lactic, succinic and other OAs has also been reported. Other fermentation products during acidogenesis are alcohols such as methanol and ethanol, carbonyl compounds, carbon dioxide and hydrogen (Grimmler et al., 2011; Li et al., 2013). Contino et al. (2011) evaluated the use of ethyl esters of low molecular weight VFAs in a homogenous charge compression ignition engine with promising results. Process parameters such as pH, temperature, the C/N ratio and hydraulic retention time play an important role, controlling the production of OAs during fermentation (Chen et al., 2013). In previous studies it was also shown that methane, alcoholic and acidogenic fermentations can be promoted in the presence of porous materials such as c-alumina pellets and kissiris (a porous volcanic rock found in Greece; 70% SiO2) acting as culture immobilization carriers (Koutinas et al., 1991; Tsoutsas et al., 1990; Galanakis et al., 2012; Syngiridis et al., 2013, 2014). The results showed that under selected process conditions (substrate concentration and pH, temperature, type of substrate) the anaerobic acidogenesis can lead to products with different compositions of OAs and ethanol, giving the possibility to produce different types of ester-based new generation biofuels (Syngiridis et al., 2013, 2014). Based on the above studies, the aim of the present study was to examine the effect of kissiris as immobilization carrier and promoter on the anaerobic acidogenesis of sucrose and sucrose/raffinose mixtures, to study the operational stability of the continuous acidogenesis of these sugars, and subsequently of vinasse, and to determine the OAs chemical composition and degree of ethanol formation.
2.4. Cell immobilization and continuous anaerobic acidogenic fermentation of sucrose The experimental apparatus used for cell immobilization and for continuous acidogenic fermentation consisted of a 1.25 L glass tower reactor placed in an incubator set at 37 °C, and connected to a high accuracy peristaltic pump. The bioreactor was filled with 950 g mineral kissiris and equal volumes of 20 g/L sucrose medium and anaerobic culture suspension. The system was left to ferment for two days at 37 °C without feeding in order to achieve cell immobilization. Subsequently, fresh sucrose medium was pumped continuously into the bioreactor and a steady state was obtained in a few days. The bioreactor was pumped continuously and successively with sucrose, sucrose and raffinose mixture and vinasse media as described below. Samples were removed at various time intervals and analyzed for OAs, ethanol and residual sugar by GC and HPLC. 2.5. Media for continuous acidogenic fermentation The various substrates that were used successively for the continuous anaerobic fermentation were: (1) 20 g/L sucrose medium for 17 days, (2) 17 g/L sucrose–3 g/L raffinose medium for 16 days, (3) 17 g/L sucrose–3 g/L raffinose–1:5 vinasse:water dilution for 8 days, (4) 1:5 vinasse:water dilution for 9 days, and finally (5) 1:10 vinasse:water dilution for 10 days. All media contained aqueous NH3 and 50% H3PO4 (COD:N:P ratio of 100:5:1), 4 g/L NaHCO3 and 4 g/L yeast extract and had pH 8. They were sterilized by autoclaving at 120 °C for 10 min. For the vinasse media only pH adjustment took place and there was no sterilization.
2. Methods
2.6. Analysis of ethanol
2.1. Culture and growth media
Ethanol was determined on a Shimadzu GC-8A system, with a Teknokroma HAYE SEP Q 80/100 column, a C-R6A Chromatopack integrator, He as carrier gas (40 mL/min), and a FID detector. The injection port and detector temperature was 210 °C. The column temperature was 130 °C. Samples of 2 mL were injected directly into the column. Determinations were done by means of standard curves.
Mixed bacterial anaerobic culture obtained from a UASB reactor (Syngiridis et al., 2013, 2014) was inoculated in a medium containing 50 g/L glucose, aqueous NH3 and 50% H3PO4 (COD:N:P ratio of 100:5:1), 4 g/L NaHCO3, 4 g/L yeast extract, and without pH adjustment (Koutinas et al., 1991). Cell growth was carried out at 37 °C. The medium was sterilized by autoclaving at 120 °C for 10 min.
2.7. Analysis of OAs 2.2. Batch acidogenic fermentation of sucrose at different pH values For the repeated batch fermentations conical flasks were used containing immobilized mixed anaerobic culture on 100 g mineral kissiris and 150 mL synthetic medium containing 40 g/L sucrose, aqueous NH3 and 50% H3PO4 (COD:N:P ratio of 100:5:1), 4 g/L NaHCO3, 4 g/L yeast extract. Fermentations were carried out at 37 °C and at different pH values from 4 to 8. The media were sterilized by autoclaving at 120 °C for 10 min. 2.3. Batch acidogenic fermentation of sucrose and raffinose mixtures For the repeated batch fermentations conical flasks were used containing immobilized mixed anaerobic culture on 100 g mineral kissiris and 100 mL synthetic medium containing 20 g/L sugars. Fermentations were carried out at 37 °C with initial pH 8. Synthetic media with five different compositions of sugars were used: (1) 20 g/L sucrose, (2) 14 g/L sucrose and 6 g/L raffinose, (3) 10 g/L sucrose and 10 g/L raffinose, (4) 6 g/L sucrose and 14 g/L raffinose, and (5) 20 g/L raffinose. Each medium also contained aqueous NH3 and 50% H3PO4 (COD:N:P ratio of 100:5:1), 4 g/L NaHCO3, and 4 g/L yeast extract. All media were sterilized by autoclaving at 120 °C for 10 min.
OAs were determined by High Performance Liquid Chromatography (HPLC), using a Jasco LC-2000 Plus chromatograph (Jasco Inc., Japan) with a Bio-rad Aminex HPX-87H column (300 7.8 mm i.d., 9 lm particle size), a PU-2089 plus quaternary gradient pump, a CO-2060 Plus oven set at 50 °C, a MD-2018 plus photodiode array detector operated at 210 nm and an AS 2050 PLUS autosampler. A solution of 0.008 N H2SO4 was used as mobile phase with a flow rate of 0.6 mL/min. The samples were filtered through disposable syringe cellulose acetate filters (CHROMAFIL) with 0.20 nm pore size and all the data were processed with ChromNav program. All determinations were done by means of standard curves. 2.8. Determination of residual sugar Residual sugar was determined by HPLC on a Shimadzu chromatograph with a NUCLEOGEL ION 300 OA column, a LC-9A pump, a CTO-10A oven at 30 °C and a RID-6A refractive index detector. H2SO4 0.008 N was used as mobile phase with a flow rate of 0.8 mL/min and propanol-1 was used as an internal standard. A volume of 0.25 mL of sample and 0.625 mL of 1% (v/v) solution of propanol-1 were diluted to 25 mL. The samples were filtered with a disposable syringe cellulose acetate filter (CHROMAFIL) with 0.20 nm pore size and then 60 lL of the final solution were injected
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131
3
0.48 ± 0.16 0.43 ± 0.07 0.42 ± 0.03 0.42 ± 0.08 0.37 ± 0.09 9.57 ± 1.11 8.34 ± 1.20 7.82 ± 0.59 7.33 ± 1.36 6.53 ± 1.57 – – – – – – 0.17 ± 0.10 – – – 1.14 ± 0.41 1.03 ± 0.25 0.92 ± 0.15 0.96 ± 0.10 1.51 ± 0.09 7.93 ± 3.56 6.39 ± 0.86 6.42 ± 0.58 6.08 ± 1.29 4.49 ± 1.46 0.11 ± 0.10 0.24 ± 0.20 0.10 ± 0.10 – 0.08 ± 0.08 – – – – – 0.39 ± 0.16 0.68 ± 0.34 0.39 ± 0.16 0.29 ± 0.09 0.45 ± 0.12 – 0.80 ± 0.26 1.20 ± 0.53 2.50 ± 0.63 2.50 ± 0.78
5.33 ± 1.00 5.67 ± 0.51 4.30 ± 0.20 5.40 ± 0.44 4.20 ± 0.53
3.23 ± 0.46 3.27 ± 0.25 3.21 ± 0.18 3.16 ± 0.02 3.52 ± 0.24 Without kissiris 100 suc 70 suc/30 raf 50 suc/50 raf 30 suc/70 raf 100 raf
6.26 ± 5.13 1.50 ± 1.06 1.00 ± 078 0.40 ± 0.17 0.37 ± 0.06
9.93 ± 1.57 10.60 ± 2.05 7.65 ± 1.69 12.44 ± 0.93 10.75 ± 0.67 4.99 ± 3.70 5.81 ± 1.94 2.86 ± 2.60 6.75 ± 0.69 5.07 ± 1.98 0.12 ± 0.10 0.23 ± 0.20 – – 0.11 ± 0.11 0.89 ± 0.06 0.94 ± 0.09 1.03 ± 0.09 0.98 ± 0.14 1.00 ± 0.09 4.48 ± 4.30 0.71 ± 0.43 3.43 ± 0.61 1.14 ± 1.02 3.49 ± 1.78 0.28 ± 0.20 0.78 ± 0.29 0.43 ± 0.10 0.50 ± 0.07 0.27 ± 0.23 0.18 ± 0.16 1.17 ± 0.92 0.20 ± 0.19 1.58 ± 0.97 0.26 ± 0.25
Propionic
With kissiris 100 suc 70 suc/30 raf 50 suc/50 raf 30 suc/70 raf 100 raf
Acetic
OAs (g/L) Residual sugar (g/L) Ethanol (mL/L) Final pH
Batch acidogenesis was performed at 37 °C and at pH 8 as the optimum conditions. Culture immobilization on kissiris increased
Substrate composition (%)
3.2. Effect of immobilization on OAs and ethanol production during acidogenesis of sucrose and raffinose mixtures
Table 1 Batch acidogenesis of sucrose (suc) and raffinose (raf) mixtures using anaerobic mixed culture immobilized on kissiris.
Since it is well established that factors such as pH and fermentation temperature control affect the production of OAs during anaerobic fermentation (Chen et al., 2013), an optimization study of these parameters took place using cells immobilized on kissiris and free cells and sucrose as substrate. In order to identify the effect of pH (4–8), repeated batch acidogenic fermentations of sucrose (40 g/L) were performed at 37 °C. At all pH values the use of kissiris led to increased OAs concentration. Specifically, the production of OAs increased with increasing pH, with the highest concentration (23 g/L) obtained at pH 8, which is in accordance with previous studies (Jiang et al., 2013; Wang et al., 2014). The main acid produced using both free and immobilized cells was lactic acid (more than 50% of total OAs) followed by succinic acid and acetic acid. A small amount of ethanol was also produced varying from 1.6 (at pH 8) to 6.5 mL/L (at pH 6) for free cells and from 1.7 (at pH 8) to 3.4 mL/L (at pH 6) for cells immobilized on kissiris. These results suggest that ethanol-type fermentation also took place. In order to evaluate the effect of temperature, fermentations were also carried out at 18–52 °C, with substrate of pH 7 for free cells and pH 8 for immobilized cells. In all cases the promotional effect of kissiris was observed leading to almost two-fold higher OAs concentrations. In the case of kissiris, an increase in OAs concentrations was observed with the increase of temperature from 18 °C to 37 °C. The OAs concentration at 52 °C (10.1 g/L) was lower than that at 37 °C (14.9 g/L), indicating a lower acidogenic activity at higher temperatures, which is in accordance to other studies (Jiang et al., 2013; Komemoto et al., 2009). The main acid was lactic acid (more than 50% of total OAs) indicating that lactate-type fermentation occurred as was also observed in other studies (Wang et al., 2014).
Butyric
3.1. Effect of pH and fermentation temperature on OAs and ethanol production during sucrose acidogenesis
0.27 ± 0.08 1.21 ± 0.59 0.66 ± 0.25 1.50 ± 0.84 0.55 ± 0.06
Lactic
Succinic
Isovaleric
Valeric
The last few years, research has focused on the production of new generation biofuels based on ester production using the simultaneously produced OAs and ethanol during acidogenic fermentation of carbohydrates (Ren et al., 1997). Furthermore, in recent studies the promotional effect of porous materials serving as culture immobilization carriers, such as c-alumina, on glucose acidogenesis was also shown (Syngiridis et al., 2013, 2014). These works open the way for the production of the new generation biofuels from agro-industrial wastes. Vinasse the liquid waste of ethanol distilleries, contains sucrose and raffinose as residual sugar. Therefore, as a first step to evaluate vinasse as substrate for esters production, the study of sucrose and raffinose acidogenesis was necessary. Mineral kissiris, a volcanic rock found in Greece, is a very cheap and abundant porous material compared to c-alumina. Therefore the objectives of this study were to investigate any promotional effects of kissiris, as compared with free cells, on the acidogenesis of sucrose and raffinose mixtures and vinasse regarding the composition of the accumulating OAs and ethanol formation in batch and continuous operation.
– – – – –
Total (g/L)
3. Results and discussion
0.17 ± 0.12 0.17 ± 0.06 0.30 ± 0.17 0.27 ± 0.06 0.20 ± 0.15
Yield (g/g)
directly to the column. Residual sugar concentrations were calculated using standard curves.
0.50 ± 0.18 0.53 ± 0.13 0.38 ± 0.12 0.62 ± 0.05 0.54 ± 0.03
K. Lappa et al. / Bioresource Technology xxx (2015) xxx–xxx
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131
4
K. Lappa et al. / Bioresource Technology xxx (2015) xxx–xxx
Fig. 1. OAs composition of the effluents produced during the continuous acidogenesis of various influents, at 37 °C and initial pH 8, using mixed anaerobic culture immobilized on kissiris.
Table 2 Continuous acidogenesis of sucrose (suc) and sucrose/raffinose (raf) media using anaerobic mixed culture immobilized on kissiris. Days (d)
Final pH
Ethanol (ml/L)
OAs (g/L) Acetic
Propionic
Isobutyric
Butyric
Lactic
Succinic
Isovaleric
Total
Yield (g/g)
Productivity (g/Ld)
0.90 0.89 0.30 0.18 1.05 3.02 0.81 0.34 0.49 0.39 0.55 0.06 0.03 1.66
0.31 0.29 0.24 0.41 0.38 0.24 0.20 0.54 0.32 0.29 0.23 0.19 – 0.21
– – – – – – – – – – – – – –
– – – – – – – – – – – – – –
– – 0.35 0.48 0.80 1.06 0.96 3.34 1.01 0.69 0.39 0.27 – –
9.18 9.16 9.68 7.01 9.88 7.58 5.21 6.58 7.00 7.94 8.90 6.74 7.98 8.34
– – – – 0.88 0.94 0.93 0.89 0.96 1.07 1.21 1.22 1.67 1.45
– – – – – – – – – – – – – 0.28
9.49 9.45 10.27 7.90 11.94 9.82 7.30 11.35 9.29 9.99 10.73 8.42 9.65 11.58
0.47 0.47 0.51 0.40 0.60 0.49 0.37 0.57 0.47 0.50 0.54 0.42 0.48 0.60
3.80 3.78 4.11 3.16 4.78 3.93 2.92 4.54 3.72 4.00 4.29 3.37 3.86 4.63
Feed: suc 17 g/L/raf 3 g/L 20 5.0 0.85 21 4.3 0.42 22 4.4 1.32 23 4.6 0.10 27 4.7 1.27 28 4.3 0.50 31 3.7 2.75 32 4.0 0.30 33 4.1 2.60
0.46 0.40 0.38 0.28 0.45 0.29 0.19 – –
0.32 0.22 – – – – – – –
1.38 1.34 1.35 1.22 1.56 1.24 – – –
– – – – – – – – –
4.03 6.26 6.49 5.77 4.98 5.61 5.26 5.48 7.57
1.19 0.91 – – 0.87 0.90 0.90 0.99 0.93
– – 0.47 0.53 – – – – –
7.38 9.13 8.69 7.80 7.86 8.04 6.35 6.47 8.50
0.37 0.46 0.43 0.39 0.39 0.40 0.32 0.32 0.43
2.95 3.65 3.48 3.12 3.14 3.22 2.54 2.59 3.40
Feed: suc 20 g/L 1 3.8 2 4.2 3 4.6 4 5.3 5 4.6 7 4.4 8 4.6 9 4.7 10 4.4 12 4.4 13 4.2 14 4.9 16 4.5 17 4.3
the OAs yield in most of the tested sucrose/raffinose mixtures as compared with free cells. The highest improvement was observed in the case of 100% raffinose and the 30% sucrose–70% raffinose mixture. Table 1 shows the results of the batch acidogenesis of sucrose and raffinose mixtures with and without the use of kissiris. Bioconversion of sucrose and raffinose occurred in all fermentation batches indicating the capability of the mixed anaerobic culture to ferment this type of sugars. The OAs yield factor varied from 0.38 to 0.62 g/g for an initial sugar concentration of 20 g/L. OAs concentration ranged from 5.9 to 13.4 g/L, while ethanol formation did not exceed 0.5 mL/L. Without kissiris the OAs concentrations and yield factors were lower, while higher concentrations of ethanol were observed. OAs that were formed were lactic, valeric, acetic, propi-
onic, succinic, and butyric acids, and in few cases isovaleric acid. Lactic acid and valeric acid were the predominant acids in most cases. These results encouraged the subsequent study of continuous acidogenesis using immobilized cells in order to achieve the aforementioned objectives. 3.3. OAs and ethanol production during continuous acidogenesis of sucrose and sucrose/raffinose mixtures Continuous acidogenesis was performed using successively sucrose, sucrose/raffinose mixtures and finally vinasse solutions (Fig. 1). During the continuous fermentation of sucrose and sucrose/raffinose mixtures (Table 2), lactic acid was produced at
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131
5
K. Lappa et al. / Bioresource Technology xxx (2015) xxx–xxx Table 3 Continuous acidogenesis of vinasse using anaerobic mixed culture immobilized on kissiris. Days (d)
Final pH
Ethanol (ml/L)
OAs (g/L) Acetic
Propionic
Isobutyric
Butyric
Lactic
Succinic
Isovaleric
1.21 1.37
– –
– –
– –
– –
4.50 5.45
– 0.81
– –
4.50 6.26
1.80 2.50
Feed: 50% vinasse 39 4.5
2.92
–
–
–
–
3.52
0.84
–
4.36
1.74
Feed: 75% vinasse 41 5.0
1.27
0.37
–
–
–
7.58
–
–
7.95
3.18
Feed: 100% vinasse (1:5) 43 5.8 44 6.6 46 6.6 47 6.7 48 6.3 49 6.2 50 6.9
1.10 – 0.71 0.80 3.12 1.04 1.32
– 3.36 3.48 3.68 2.91 3.19 2.60
– 1.30 – 0.97 – – –
– 1.11 1.63 1.52 1.52 1.51 1.34
3.19 7.56 7.74 8.65 7.45 8.52 7.79
2.44 – – – – – –
– – – – – – –
– – – 0.51 – – –
5.64 13.33 12.85 15.33 11.88 13.22 11.73
2.26 5.33 5.14 6.13 4.75 5.29 4.69
Feed: 100% vinasse (1:10) 53 6.8 1.35 54 7.9 0.10 55 7.9 2.07 57 7.8 0.34 60 6.7 0.30
1.12 0.99 1.59 0.87 1.66
– – – – –
– – – – –
2.40 3.10 4.02 4.13 3.07
– – – – –
– – – – –
– – – – –
3.52 4.09 5.61 5.00 4.73
1.41 1.64 2.24 2.00 1.89
Feed: 25% vinasse 35 4.1 37 3.9
higher amounts compared with the other acids formed (acetic, propionic, isobutyric, butyric, succinic and isovaleric). The total OAs concentration varied between 6.4 and 12 g/L. Lactic acid accounted for about 80% of total OAs while butyric acid production was very low and occurred only during the first 14 days of operation. There was no butyric acid production observed when sucrose–raffinose mixtures were used. Ethanol production was low (up to 3 mL/L). The sugar conversion was 100%. 3.4. OAs production during continuous acidogenesis of vinasse After more than a month of operation, vinasse was used as influent in the continuous system and the results are summarized in Table 3. Firstly a dilution of concentrated vinasse 1:5 with water was used for 9 days simulating the content of fresh vinasse. The use of vinasse caused an increase on the total OAs content, which reached 15.3 g/L and also changed their composition (Table 3 and Fig. 1). The lactic acid concentration decreased instantly and subsequently butyric acid and acetic acid concentration increased to 7.3 and 2.7 g/L, respectively. Butyric acid accounted for 60% of total OAs and acetic acid for 23%. Ethanol production was not affected and concentrations up to 3.1 mL/L were reported. The operation was continued for ten more days using a 1:10 dilution ratio of concentrated vinasse with water (accounting for a 1:2 dilution of fresh vinasse). This led to a decrease in total OAs to a mean value of 4.6 g/L, while only butyric and acetic acids were produced. 3.5. Discussion Mineral kissiris is a volcanic siliceous and porous material of very low cost. In various previous studies it has been indicated as a suitable material for use as cell immobilization carrier and promoter of alcoholic fermentation processes (Kourkoutas et al., 2004). Therefore, the idea was to further examine this material as promoter of the acidogenesis of vinasse. The effect of pH and temperature conditions were initially studied, showing that the best pH was 8 and the optimum temperature 37 °C, leading to an 80% improvement of yield when kissiris was used. These results encouraged the study of continuous acidogenesis of vinasse (Tables 2 and 3). However, due to the poor results obtained when vinasse
Total
Productivity (g/Ld)
was used directly, adaptation of the culture was done using successively sucrose, sucrose/raffinose mixtures, sucrose/raffinose/vinasse mixtures and finally vinasse. This adaptation process led to a substantial improvement of the acidogenesis of vinasse with an operational stability of at least two months. Table 3 shows that the predominant acid was butyric acid and the ethanol which was formed simultaneously had an average concentration of 1 mL/L, suggesting that it could esterify about the 15% of the produced OAs. In order to cover the amount of ethanol required for esterification of the total amount of OAs, the study of a system consisting of an additional bioreactor containing immobilized Saccharomyces cerevisiae and fed with carbohydrate containing wastes is proposed. The treatment of vinasse led to a 15 g/L OAs concentration, which could give about 25 mL/L esters. Suggesting that the esterification would be performed in a non-polar organic solvent using lipases, the production of an ester-based biofuel could be done with low energy demand. Because esters are non-polar and can form a second layer on the aqueous phase, their separation could also be done with low energy consumption when they are produced by chemical esterification. 4. Conclusions The use of kissiris as mixed anaerobic culture immobilization carrier promoted the batch and continuous acidogenic fermentation of sucrose and raffinose mixtures. The system presented high operational stability, and the successful adaptation of the biocatalyst in sucrose/raffinose media resulted to the possibility for scale-up of the acidogenesis of vinasse. Lactic acid-type fermentation occurred in synthetic sugar media, while butyric acid-type fermentation occurred when vinasse was used. The acidogenesis of vinasse in the presence of kissiris and after an adaptation treatment is promising for a new generation ester-based biofuel technology development. Acknowledgements Research project co-financed by the European Union (European Regional Development Fund – ERDF) and Greek National Funds through the Operational Program ‘‘Competitiveness and
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131
6
K. Lappa et al. / Bioresource Technology xxx (2015) xxx–xxx
Entrepreneurship’’ of the National Strategic Reference Framework (NSRF) 2007–2013 – National Action ‘‘Cooperation 2011: Partnerships of Production and Research Institutions in Focused Research and Technology Sectors’’ of General Secretariat for Research and Technology (Contract Nr. 11SYN-8-839). References Balat, M., Balat, H., 2009. Recent trends in global production and utilization of bioethanol fuel. Appl. Energy 86, 2273–2282. Chen, H., Meng, H.J., Nie, Z.C., Zhang, M.M., 2013. Polyhydroxyalkanoate production from fermented volatile fatty acids: effect of pH and feeding regimes. Bioresour. Technol. 128, 533–538. Contino, F., Foucher, F., Mounaim-Rousselle, C., Jeanmart, H., 2011. Combustion characteristics of tricomponent fuel blends of ethyl acetate, ethyl propionate, and ethyl butyrate in homogeneous charge compression ignition (HCCI). Energy Fuel 25, 1497–1503. De La Rubia, M.A., Raposo, F., Rincón, B., Borja, R., 2009. Evaluation of the hydrolytic–acidogenic step of a two-stage mesophilic anaerobic digestion process of sunflower oil cake. Bioresour. Technol. 100, 4133–4138. Eggeman, T., Verser, D., 2005. Recovery of organic acids from fermentation broths. Appl. Biochem. Biotechnol. 121–124, 605–618. España-Gamboa, E.I., Mijangos-Cortés, J.O., Hernández-Zárate, G., Maldonado, J.A.D., Alzate-Gaviria, L.M., 2012. Methane production by treating vinasses from hydrous ethanol using a modified UASB reactor. Biotechnol. Biofuels 5, 82. Fontanille, P., Kumar, V., Christophe, G., Nouaille, R., Larroche, C., 2012. Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Yarrowia lipolytica. Bioresour. Technol. 114, 443–449. Galanakis, C.M., Kordulis, C., Kanellaki, M., Koutinas, A.A., Bekatorou, A., Lycourghiotis, A., 2012. Effect of pressure and temperature on alcoholic fermentation by Saccharomyces cerevisiae immobilized on c-alumina pellets. Bioresour. Technol. 114, 492–498. Goldemberg, J., Coelho, S.T., Guardabassi, P., 2008. The sustainability of ethanol production from sugarcane. Energy Policy 36, 2086–2097. Grimmler, G., Janssen, H., Krauße, D., Fischer, R.-J., Bahl, H., Dürre, P., Liebl, W., Ehrenreich, A., 2011. Genome-wide gene expression analysis of the switch
between acidogenesis and solventogenesis in continuous cultures of Clostridium acetobutylicum. J. Mol. Microbiol. Biotechnol. 20, 1–15. Jiang, J., Zhang, Y., Li, K., Wang, Q., Gong, C., Li, M., 2013. Volatile fatty acids production from food wastes: Effects of pH, temperature, and organic loading rate. Bioresour. Technol. 143, 525–530. Komemoto, K., Lim, Y.G., Nagao, N., Onoue, Y., Niwa, C., Toda, T., 2009. Effect of temperature on VFAs and biogas production in anaerobic solubilization of food waste. Waste Manag. 29, 2950–2955. Kourkoutas, Y., Bekatorou, A., Banat, I.M., Marchant, R., Koutinas, A.A., 2004. Immobilization technologies and support materials suitable in alcohol beverages production: a review. Food Microbiol. 2, 377–397. Koutinas, A.A., Toutoutzidakis, G., Kana, K., Kouinis, I., 1991. Methane fermentation promoted by gamma-alumina pellets. J. Ferment. Bioeng. 72, 64–67. Li, Y., Hua, D., Zhang, J., Zhao, Y., Xu, H., Liang, X., Zhanq, X., 2013. Volatile fatty acids distribution during acidogenesis of algal residues with pH control. World J. Microb. Biotechnol. 29, 1067–1073. Navarro, A.R., Sepúlveda, M.del C., Rubio, M.C., 2000. Bio-concentration of vinasse from the alcoholic fermentation of sugar cane molasses. Waste Manag. 20, 581– 585. Ren, N.Q., Wang, B.Z., Huang, J.C., 1997. Ethanol-type fermentation from carbohydrate in high rate acidogenic reactor. Biotechnol. Bioeng. 54, 428–433. Syngiridis, K., Bekatorou, A., Kallis, M., Kandylis, P., Kanellaki, M., Koutinas, A.A., 2013. c-Alumina as a process advancing tool for a new generation biofuel. Bioresour. Technol. 132, 45–48. Syngiridis, K., Bekatorou, A., Kandylis, P., Larroche, C., Kanellaki, M., Koutinas, A.A., 2014. Favouring butyrate production for a new generation biofuel by acidogenic glucose fermentation using cells immobilised on c-alumina. Bioresour. Technol. 161, 118–123. Tsoutsas, T., Kanellaki, M., Psarianos, C., Kalliafas, A., Koutinas, A.A., 1990. Kissiris-a mineral support for the promotion of ethanol fermentation by Saccharomycescerevisiae. J. Ferment. Bioeng. 69, 93–97. Wang, K., Yin, J., Shen, D., Li, N., 2014. Anaerobic digestion of food waste for volatile fatty acids (VFAs) production with different types of inoculum: Effect of pH. Bioresour. Technol. 161, 395–401. Wheals, A.E., Basso, L.C., Alves, D.M.G., Amorim, H.V., 1999. Fuel ethanol after 25 years. Trends Biotechnol. 17, 482–487.
Please cite this article in press as: Lappa, K., et al. Continuous acidogenesis of sucrose, raffinose and vinasse using mineral kissiris as promoter. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.01.131