Lactic acid production from acidogenic fermentation of fruit and vegetable wastes

Bioresource Technology 191 (2015) 53–58

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Lactic acid production from acidogenic fermentation of fruit and vegetable wastes Yuanyuan Wu, Hailing Ma, Mingyue Zheng, Kaijun Wang ⇑ State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing 100084, China

h i g h l i g h t s  Lactic acid with amount of 10–20 g/L was stably produced at pH 4.0.  The fermentation type was finally converted into heterofermentation at pH 4.0.  Hydrolysis was enhanced and lactic acid fermentation was improved at pH 5.0.  Bifidobacterium played an important role for lactic acid production at pH 5.0.

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Article history: Received 19 March 2015 Received in revised form 23 April 2015 Accepted 25 April 2015 Available online 7 May 2015 Keywords: Lactic acid Heterofermentation Anaerobic digestion Acidogenic fermentation Fruit and vegetable wastes

a b s t r a c t This work focused on the lactic acid production from acidogenic fermentation of fruit and vegetable wastes treatment. A long term completely stirred tank reactor (CSTR) lasting for 50 days was operated at organic loading rate (OLR) of 11 gVS/(L d) and sludge retention time (SRT) of 3 days with pH controlled at 4.0 (1–24 day) and 5.0 (25–50 day). The results indicated that high amount of approximately 10–20 g/L lactic acid was produced at pH of 4.0 and the fermentation type converted from coexistence of homofermentation and heterofermentation into heterofermentation. At pH of 5.0, the hydrolysis reaction was improved and the total concentration of fermentation products increased up to 29.5 gCOD/L. The heterofermentation was maintained, however, bifidus pathway by Bifidobacterium played an important role. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction In China, fruit and vegetable wastes (FVWs) are greatly generated approximately 1.3 million tons per day in Chinese cities, however, less than 20% were properly treated (Shen et al., 2013; Liu et al., 2012). Anaerobic digestion has been considered to be the most promising alternative technology for FVWs treatment. With the characteristics of high volatile solids and easy degradability, the FVWs could be rapidly hydrolyzed, which might lead to the acid accumulation and inhibition to methanogenesis if the reactor is overloaded. Ganesh et al. (2014) summarized the published literatures and concluded that the maximum OLR for single-phase anaerobic digestion of FVWs was within 3.6 KgVS/(m3 d). According to the study of Shen et al. (2013), volatile fatty acids (VFAs) would accumulate to 2.3 g/L and pH would decrease to 6.8 if the OLR was increased to 3.5 KgVS/(m3 d). And then, two-phase anaerobic digestion technology was applied for FVWs ⇑ Corresponding author. Tel.: +86 10 62789411; fax: +86 10 62773065. E-mail address: [email protected] (K. Wang). http://dx.doi.org/10.1016/j.biortech.2015.04.100 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

treatment to keep the stability of whole system at high OLR (Mtz-Viturtia et al., 1995; Bouallagui et al., 2004; Shen et al., 2013). However, the OLR of two-phase anaerobic digestion were only reported with limited increase to 5.7–7.7 KgVS/(m3 d), which was summarized by Ganesh et al. (2014). In addition, the construction and operational cost were also increased for the additional reactors and alkaline addition. Recently the concept of VFAs platform has been proposed and based on that, several end products other than methane such as polyhydroxyalkanoates (PHAs), medium chain fatty acids, biofuels or hydrogen gas are being researched (Chang et al., 2010; Reis et al., 2003; Agler et al., 2012; Steinbusch et al., 2000). There have been a lot of researches on the VFAs production from different wastes or wastewaters. Jiang et al. (2013) found that the optimal condition for VFAs production were pH 6.0, 35 °C, 11gTS/(L d) from food waste with acetate and butyrate accounting for 60%. Chen et al. (2007) reported that alkaline pH (8.0–11.0) could significantly improve the total VFAs concentration from waste activated sludge and acetate, propionate and isovaleric acid were the three main VFAs. Yu and Fang (2002) recommended that productions

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of butyrate and acetate were favored at pH of 6.0–6.5 while ethanol and propionate at pH 4.0–4.5 from dairy wastewater. However, most of the studies focused on VFAs production (acetate, propionate, iso-butyrate, butyrate, iso-valerate, valerate), only a handful of studies paid attention to other fermentation products such as lactic acid, ethanol or succinate. Lactic acid has been verified as an available building block for PHAs and it is also widely used in industries such as the pharmaceutical, biomaterial, detergent, leather, and textile industries (Jiang et al., 2011; Kim et al., 2012; Mazzoli et al., 2014). Temudo et al. (2007) investigated the influence of pH on glucose fermentation by mixed culture and indicated that lactic acid with low yield rate of 0.2 mol/mol glucose was produced at pH of 4.0–5.0. Wang et al. (2014) evaluated the effect of pH on acidogenic end products from food waste in batch experiments and found that high concentration of 18 g/L lactic acid was produced at pH of 4.0. Selective production of lactic acid was tried with glucose as substrate by Itoh et al. (2012) and results showed that lactic acid was produced at low pH of 3.5, however, the lactic fermentation was not stable. Thus, the lactic acid could be produced from the acidogenic fermentation at low pH, however further investigation such as the fermentation stability in long term operation, fermentation mechanism and bacterial community are still necessary. Therefore, a long term CSTR reactor was operated to investigate the possibility and stability of lactic acid production from acidogenic fermentation of FVWs treatment. The bacterial community and fermentation mechanism for lactic acid fermentation were also investigated. In addition, the method of increasing pH was also tried to improve the lactic acid fermentation.

2. Methods

2.3. Analytical methods The production of gas was flowed to the gas counters manufactured by Bioprocess AB, Lund, Sweden. Gas samples were taken through the sampling port located on the top of the reactor while effluent samples were collected from the draw-off materials every day. TS, VS, TSS and VSS were measured according to the standard methods (APHA, 1998). The tCOD and sCOD were determined by spectrophotometry (DR6000, HACH Company, Germany) and sCOD was measured after filtration (0.45 lm). The total COD of fermentation products marked as tCODp was calculated by the sum of COD of individual fermentation product. Volatile fatty acids (acetate, propionate, butyrate, iso-butyrate, valerate, iso-valerate) and other fermentation products including caproate, lactate, succinic acid and formic acid were determined by high performance liquid chromatography (SHIMADZU Company, Japan), using an Aminex HPX-87H column (T = 50 °C) from Bio Rad coupled to an UV (210 nm) detector, while sulfuric acid 5 mM was used as eluent at a rate of 0.5 ml/min. Ethanol was determined by gas chromatography (Agilent 7890A, Agilent Technologies, America) equipped with a capillary column (DB-FFAP, 30 m  0.53 mm  1 lm) and a flame ionization detector. The temperature of the injector and detector were both 230 °C. The column temperature was increased from 70 °C to 180 °C at a rate of 20 °C/min and kept at 180 °C for an additional 5 min. The nitrogen gas was used as carrier gas. Gas content (hydrogen gas, carbon monoxide, carbon dioxide and methane) was determined by gas chromatograph (Agilent, 7890A) fitted with a thermal conductivity detector and Agilent Carboxen 1000 column (4.5 m  2 mm; mesh size 60/80). The argon gas was used as carrier gas with a rate of 30 mL/min. The temperature of injector, detector and column were 150 °C, 250 °C and 150 °C, respectively.

2.1. Inoculums and substrate 2.4. Bacterial community analysis The seed sludge was taken from anaerobic digester in Xiao Hongmen wastewater treatment plant in Beijing city, China. The pH, total suspended solids (TSS) and volatile suspended solids (VSS) concentration of the sludge were 7.8, 8.66 g/L, and 4.86 g/L, respectively. Simulated FVWs was used as substrate composing 57% watermelon, 29% apple and 14% potato by wet weight. After crushed by a food waste disposer (produced by In Sink erator company, Model 55, 410w) and fully mixed, the simulated FVWs was stored at 20 °C in a refrigerator and the frozen FVWs was thawed at 4 °C before use every day. Raw shredded FVWs were analyzed more than ten times, and the initial total solids (TS) concentration and volatile solids (VS) were approximately 100 g/kg and 87 g/kg, respectively. The total COD (tCOD), soluble COD (sCOD) and NH+4-N were 137.1, 79.3 and 0.07 g/kg, respectively, and the pH was 4.5–4.8.

2.2. Reactors operation design A CSTR reactor with a working volume of 1.5 L (total 2 L) was operated at a greenhouse maintained at 35 °C. The reactor was fully stirred by magnetically stirring. The SRT of the CSTR reactor was set at 3 days and OLR was 11 gVS/(L d) by feeding diluted FVWs (200 g raw FVWs diluted to 500 ml with tap water). The pH of CSTR reactor was controlled at 4.0 (stage 1: 1–24 day) and 5.0 (stage 2: 25–50 day) by a pH controller with addition of HCl (2 M) and NaOH (2 M). The reactor was fed and drawn off once a day and the draw-off effluent was used for analysis. The characteristics of effluent were determined every day in the first 24 days and every two days in the 25–50 day.

2.4.1. Clone library analysis To analyze the structure of bacterial communities in the CSTR, the sludge of the 24th day in the CSTR was used for clone library analysis and high throughput pyrosequencing. In addition, the sludge of the 50th day was used for high throughput pyrosequencing. The deoxyribonucleic acids (DNAs) of different sludge samples were extracted using the Fast DNA SPIN Kit for Soil (MP Biomedicals LLC., Califonia, USA). The primers 27F (50 -AGAGTTTGA TCCTGGCTCAG-30 ) and1522R (50 -AAGGAGGTGATCCAGCCAGCCGC A-30 ) were used for the amplification of bacterial 16S rRNA genes (Fuller et al., 2003). The polymerase chain reaction (PCR) was performed in a final volume of 50 lL containing 5 lL of DNA buffer, 4 lL of deoxy-ribonucleoside triphosphate (dNTP), 0.5 lL of each primer, 1 lL of extracted DNA solutions, 0.5 lL Taq DNA polymerase and 38.5 lL sterile water. The operations of amplification of bacterial 16S rRNA genes were as follows: 94 °C for 2 min, 30 cycles of 94 °C for 1 min, annealing at 50 °C for 1 min and extension at 72 °C for 1.5 min and final extension at 72 °C for 5 min. The PCR products were purified with QIA quick PCR purification kit (Qiagen Company, Hilden, Germany) and then cloned into PMD18-T vector (Takara Biotechnology Co., Ltd., Dalian, China). 50 Positive clones were sequenced (completed by Takara Biotechnology Co., Ltd., Dalian, China), which were randomly chosen using a 3730XLDNAAnalyzer (Applied Biosystems Company, USA) and all sequences were submitted to the BLASTN (NCBI, USA) to obtain the closest relatives. 2.4.2. High throughput pyrosequencing and sequence analysis The samples were sent to GENEWIZ, Inc. (Jiangsu, China) for DNA extraction and sequencing on the Illumina MiSeq

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pyrosequencing platform. After primers was removed, the raw sequences was demultiplexed, qualified and then clustered to operational taxonomical units (OTUs) except that the initial removal of sequences was aimed at quality score <25. Total of 255980 (the sample in the 24th day) and 85514 (the sample in the 50th day) high-quality 16S rRNA gene sequences were obtained with average length larger than 400 bp, but less than 480 bp.

acidogenic bacteria. Jiang et al. (2013) reported that the VFAs accounted for 6.6% at pH uncontrolled around 3.0 for food waste treatment in batch experiment. Dog˘an and Demirer (2009) found that the ratio of acid produced to sCOD was approximately 19.1% at pH uncontrolled for municipal solid waste treatment. However, in this study the concentration of tCODp was 23.5 gCOD/L and then the ratio of tCODp/sCOD was high to 84.1%. Wang et al. (2014) found that the maximum VFAs concentrations at pH 4.0 was only 2.9 gCOD/L, however, high concentration of 18.50 g/L lactic acid was also produced at pH of 4.0. Lactic acid with concentration of approximately 9.0 g/L was also found at uncontrolled pH in batch experiment (Kim et al., 2003). Therefore, lactic acid was an indispensable fermentation product at pH of 4.0 for FVWs treatment and the acidogenic bacteria still maintained relatively high activity at low pH of 4.0.

3. Results and discussion 3.1. Lactic acid fermentation In stage 1, the pH in the CSTR reactor was controlled between 4.0 ± 0.1 by the pH controller lasting for 24 days. As shown in Fig. 1, the total COD concentration of fermentation products increased gradually from start-up to 7th day and then maintained relatively stable around 23.5 ± 2.5 gCOD/L till the end of stage 1. In the stable period, lactic acid, acetate and ethanol were the three main products. In addition, lactic acid was produced with high amount of 10–20 g/L. Obviously, the concentration of lactic acid from the 16th day to 23th day was lower than the concentration from the 7th day to 15th day, however, for ethanol it was exactly on the contrary. Therefore, the whole operational time was separated into three phases: phase 1 (1–7 day), phase 2 (7–15 day), phase 3(16–24 day). The results of gas production rate and gas content during the operation of stage 1 were shown in Fig. 2. It is obvious that the gas production rate was higher in phase 3 than phase 2. The hydrogen gas, carbon monoxide, carbon dioxide and methane were determined in this study. As shown in Fig. 2, carbon dioxide took the largest proportion of more than 99% and hydrogen gas less than 1% was produced.

3.1.2. Bacterial community To reveal the bacterial community’s structure of the lactic acid fermentative CSTR, mixed liquor sample in 24th day was used for high through pyrosequencing and 16S rRNA clone library. According to the results of the high through pyrosequencing, genus Lactobacillus was observed with the largest proportion of 94.2% (Fig. 3). In addition, according to the results of clone library analysis, the Lactobacillus bacteria took a proportion of 72% and other bacteria uncultured were with a proportion of 28% (Fig. 4). Both the results of pyrosequencing and clone library indicated that the Lactobacillus was the dominant bacteria in the CSTR reactor. According to the literatures, Lactobacillus contains various different species (Hove et al., 1999). The main Lactobacillus in this study was Lactobacillus points (32%), Lactobacillus frumenti (10%), Lactobacillus acidophilus (8%) and Lactobacillus amylovorus (6%) (Fig. 4).

concentration of fermentation products(mg/l)

3.1.1. Hydrolysis and acidogenesis The average concentration of tCOD and sCOD of effluent in stage 1 were 40.7 g/L and 27.9 g/L, while the value of TS, VS, TSS, VSS and NH+4-N were 26.6 g/L, 18.9 g/L, 12.0 g/L, 10.3 g/L and 68.3 mg/L. The ratio of tCODp to sCOD is important as it indicates the activity of

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3.1.3. Fermentation type According to literatures, there are two major pathways of lactic acid production occurring within lactic acid bacteria: homofermentation and heterofermentation. Fermentation products of homofermentation are exclusively lactic acid, while heterofermentation was with equimolar amounts of CO2, lactate and acetate or ethanol except bifidobacteria (Kandler, 1983). Bifidobacteria was

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of obligately heterofermentation and obligately homofermentation took proportion of 69% and 25%, respectively, while the left 6% was facultatively heterofermentation. The average concentrations of lactic acid, ethanol and acetate in phase 2 were 191.3, 37.0 and 42.3 mM, while in phase 3 were 114.8, 86.9 and 43.7 mM. The ratio of lactic acid to the sum of ethanol and acetate in phase 2 was 2.4 while in phase 3 was 0.9. Therefore, the fermentation type in phase 2 may be the coexistence of heterofermentation and homofermentation, however, heterofermentation was very likely to be the dominant fermentation route in phase 3. In addition, the ratio of acetate to ethanol was 0.5, meaning that the heterofermentation was mainly by fermentation with equimolar amounts of CO2, lactate and ethanol. As shown in Fig. 2, the gas production rate was between 140– 250 NmL/d in phase 2 and between 300–760 NmL/d in phase 3. The gas production rate could be considered as approximately the carbon dioxide production rate due to carbon dioxide took the proportion of more than 99%. The carbon dioxide increased from phase 2 to phase 3, which was in accordance with the conversion of fermentation type from coexistence of homofermentation and heterofermentation to heterofermentation. 3.2. Improvement of lactic acid fermentation

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certified via the ‘bifidus pathway’ to produce 1.5 molecules of acetate and one molecule of lactic acid per molecule of glucose. The fermentation types of lactobacillus involved in this study were summarized according to Holzapfel and Wood (2014). The bacteria

In order to improve the lactic acid fermentation, the pH of the CSTR reactor was adjusted to 5.0 from the 25th day and the concentrations of fermentation product were shown in Fig. 5. The total COD concentration of fermentation products increased gradually from the 25th to 31th day and then maintained relatively stable around 32.1 ± 4.8 gCOD/L from the 31th to 49th day. Therefore, the total concentration of fermentation products was improved compared with stage 1. For individual fermentation product, lactic acid was produced with high amount of around 15.0 g/L. And the concentration of ethanol was between 0.8 and 1.5 g/L while acetate was approximately between 2.5–10.0 g/L. In addition, the gas volume was increased to 637.5 NmL/d and carbon dioxide was still with high average proportion of 99.2%. 3.2.1. Hydrolysis and acidogenesis The average concentration of tCOD and sCOD of effluent in the stable period of stage 2 were 43.7 and 35.0 g/L. Therefore, the ratio

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of sCOD/tCOD was 80.0%, which was obviously higher than 68.6% when in stage 1 with pH controlled at 4.0. It indicated that the hydrolysis reaction was obviously improved with the increase of pH from 4.0 to 5.0. Bouallagui et al. (2004) also reported that the optimal pH for better hydrolytic bacteria activity was between 5.0 and 6.0. The average concentration of total COD of individual fermentation product was 29.5 gCOD/L and thus the ratio of tCODp/sCOD was 84.5%, which was almost same with the ratio of 84.1% in stage 1. Therefore, the improvement of lactic acid fermentation was mainly because of the enhancement of hydrolysis reaction. 3.2.2. Bacterial community As shown in Fig. 3, the bacterial community in stage 2 was more diversity than in stage 1. Lactobacillus was still the most abundant (45.7%), followed by Bifidobacterium (22.3%), Prevotella (17.4%) and Aeriscardovia (1.2%). The genus Bifidobacterium is traditionally considered to be part of the lactic acid bacteria forming acetate and lactic acid in 3:2 molar ratio (Holzapfel and Wood, 2014). The acid susceptibility of Bifidobacterium is dependent on the strain, however, in general, it can be considered that bifidobacteria have a weak acid tolerance (Maus and Ingham, 2003). Rasic and Kurmann (1983) reported that the numbers Bifidobacterium rapidly declined at pH < 4.3. Therefore, the increase of pH to 5.0 benefited the growth of Bifidobacterium. Prevotella were reported to be able to grow at pH as low as 5.0–5.5 and could utilize glucose to produce organic acids such as formic acid, acetate, fumaric acid succinic acid (Takahashi, 2003). Aeriscardovia as a kind of Bifidobacteriaceae was a Gram-positive, non-sporeforming bacteria which degrades various carbohydrates to form acids at pH > 4.2 (Simpson et al., 2004). Therefore, the increase of pH resulted in the diversity of bacterial community, however, lactic acid bacteria including Lactobacillus and Bifidobacterium was still the dominant with the proportion of 68.0%. 3.2.3. Fermentation type A handful of butyrate was produced from the 35th day which was very likely due to the appearance of Prevotella and Aeriscardovia. Lactic acid, acetate and ethanol still took the largest

amount of more than 96% (based on mol concentration). The average concentration of lactic acid, ethanol and acetate were 172.8, 28.4 and 131.1 mM. Thus, heterofermentation was still the dominant fermentative route when pH was increased to 5.0. In addition, the ratio of acetate to ethanol changed from 0.5 at phase 3 of stage 1 to 5.0 at stage 2. The high amount of Bifidobacterium with ‘bifidus pathway’ fermentation in the CSTR may be responsible for the greatly increase of the ratio of acetate to ethanol. 4. Conclusions At pH 4.0, lactic acid with amount of 10.0–20.0 g/L was stably produced from the FVWs treatment. The fermentation type was converted from coexistence of homofermentation and heterofermentation into heterofermentation. At pH 5.0, the hydrolysis reaction was enhanced and thus the lactic acid fermentation was improved. The heterofermentation was maintained as dominant fermentation type, however, bifidus pathway by Bifidobacterium played an important role. Acknowledgements This work was financially supported by National Natural Science Foundation of China (21206084) and Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1152). References APHA, 1998. Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, Washington, DC. Agler, M.T., Spirito, C.M., Usack, J.G., Werner, J.J., Angenent, L.T., 2012. Chain elongation with reactor microbiomes: upgrading dilute ethanol to mediumchain carboxylates. Energy Environ. Sci. 5, 8189–8192. Bouallagui, H., Torrijos, M., Godon, J.J., et al., 2004. Two-phase anaerobic digestion of fruit and vegetable wastes: bioreactors performance. Biochem. Eng. J. 21, 193– 197. Chen, Y.G., Jiang, S., Yuan, H.Y., Zhou, Q., Gu, G.W., 2007. Hydrolysis and acidification of waste activated sludge at different pHs. Water Res. 41, 683–689. Chang, H., Kim, N., Kang, J., Jeong, C., 2010. Biomass-derived volatile fatty acid platform for fuels and chemicals. Biotechnol. Bioprocess Eng. 15, 1–10.

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