Journal of Environmental Sciences 2011, 23(6) 1020–1026

Application of waste frying in the biosynthesis of biodemulsifier by a demulsifying strain Alcaligenes sp. S-XJ-1

Jia Liu1, Kaiming Peng2, Xiangfeng Huang2,∗, Lijun Lu2, Hang Cheng2, Dianhai Yang2, Qi Zhou2, Huiping Deng2

1. Tongji University, Shanghai 200092, China. E-mail: [email protected] 2. College of Environmental Science & Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai 200092, China.

Received 13 October 2010; revised 03 January 2011; accepted 05 January 2011

Abstract Exploration of biodemulsifiers has become a new research aspect. Using waste frying oils (WFOs) as carbon source to synthesize biodemulsifiers has a potential prospect to decrease production cost and to improve the application of biodemulsifiers in the oilfield. In this study, a demulsifying strain, Alcaligenes sp. S-XJ-1, was investigated to synthesize a biodemulsifier using waste frying oils as carbon source. It was found that the increase of initial pH of culture medium could increase the biodemulsifier yield but decrease the demulsification ratio compared to that using paraffin as carbon source. In addition, a biodemulsifier produced by waste frying oils and paraffin as mixed carbon source had a lower demulsification capability compared with that produced by paraffin or waste frying as sole carbon source. Fed-batch fermentation of biodemulsifier using waste frying oils as supplementary carbon source was found to be a suitable method. Mechanism of waste frying oils utilization was studied by using , olein and tristearin as sole carbon sources to synthesize biodemulsifier. The results showed saturated long-chain was difficult for S-XJ-1 to utilize but could effectively enhance the demulsification ability of the produced biodemulsifier. Moreover, FT-IR result showed that the demulsification capability of biodemulsifiers was associated with the content of C=O group and nitrogen element. Key words: waste frying oils; biodemulsifier; fatty acid ; Alcaligenes sp. DOI: 10.1016/S1001-0742(10)60508-6 Citation: Liu J, Peng K M, Huang X F, Lu L J, Cheng H, Yang D H et al., 2011. Application of waste frying oils in the biosynthesis of biodemulsifier by a demulsifying strain Alcaligenes sp. S-XJ-1. Journal of Environmental Sciences, 23(6): 1020–1026

Introduction Lee, 2000). However, the prices of these hydrocarbon are higher than the water-soluble carbon sources. In the oil exploration industry, large amount of demul- In recent years, various agriculture and industry wastes sifiers is in great need for demulsification of the crude are employed as substrates to produce biosurfactants to oil emulsion. Compared to conventional chemical demul- decrease the cost, such as curd whey (Dubey and Juwarkar, sifiers, biodemulsifiers are characterized by following 2001; Joshi et al., 2008), potato process effluents (Thomp- traits: low toxicity, environmental compatibility, and high son et al., 2000; Noah et al., 2002; Thompson et al., 2001), demulsifying efficiency in extreme conditions. However, rice straw decomposing (Zhang et al., 2008), cashew biodemulsifiers have not been applied in the petroleum apple juice (Rocha et al., 2006), orange peelings (George industry due to that their production cost is higher than and Jayachandran, 2009) and waste frying oils (WFOs). chemical demulsifiers. The cost of culture medium con- Among these substrates, waste frying oils (WFOs) pro- tributes a large proportion to the high production cost of the duced from food and beverage industry are widely studied biodemulsifier. It is estimated that culture media accounts (de Lima et al., 2009). Most of these work focus on the for 30%–50% of the production cost of biosurfactants effect of WFOs on the yield, surface activity and emul- (Bednarski et al., 2004; Thavasi et al., 2007), in which cost sification capability of biosurfactant (Felse et al., 2007; of carbon source is the highest. For most of the demulsify- Rufino et al., 2007; Nitschke et al., 2005). Several studies ing strains, hydrophobic materials are preferred as carbon showed that WFOs are potential appropriate substrate, source to synthesize biodemulsifier, such as cetane (Cairns which not only increase the yield of glycolipid but also et al.,1982; Stewart et al.,1983), tetradecane (Das, 2001), dramatically decrease the production cost (Bednarsk et al., crude oils (Nadarajah et al., 2002), and kerosene (Lee and 2004; de Lima et al., 2009). However, it is rarely seen the relevant report on producing a biodemulsifier using WFOs. * Corresponding author. E-mail: [email protected] jesc.ac.cn No. 6 Application of waste frying oils in the biosynthesis of biodemulsifier by a demulsifying strain Alcaligenes sp. S-XJ-1 1021

Therefore, it is essential to investigate the synthesis of a 1.3 Biodemulsifier yield biodemulsifier in the use of WFOs as carbon source. To evaluate the biodemulsifier yield, 20 mL of carbon In previous work, one high efficient demulsifying strain tetrachloride was firstly added to 50 mL of fermented Alcaligenes sp. S-XJ-1 was isolated and intensively studied broth, and then mixed three times for extracting the on its demulsification ability. Biodemulsifier produced at residual carbon source. After undisturbed for 20 min, pH 7 with paraffin as carbon source achieved 95% of the water-pellet phase and carbon tetrachloride phase was demulsification ratio but with a lower yield of 1.57 g/L. separated. Then, the water-pellet phase was centrifuged for This study aimed to develop an appropriate method of us- 10 min at 12,000 r/min. The obtained cell pellets were thor- ing WFOs as carbon source to enhance the production and oughly cleansed with distilled water and then lyophilized demulsification ability of a biodemulsifier. Effect of fatty at –50◦ for 24 hr. The freeze dried-powder product was acid on the biosynthesis of biodemulsifiers was weighed and calculated to identify the biodemulsifier also studied to reveal the mechanism of WFOs utilization. yield. 1 Materials and methods 1.4 Emulsion preparation and demulsification test Water-in-oil model emulsion was prepared according to 1.1 Microorganism and medium the protocol introduced by Liu et al. (2009). Distilled water containing 1.9% (W/V) Span 80 and aviation kerosene The tested strain, Alcaligenes sp. S-XJ-1, was isolated containing 0.1% (W/V) Tween 80 were mixed at a volume from petroleum-contaminated soil. The liquid medium for ration of 3:2 at 10,000 r/min for 3.5 min with the aid of enrichment consisted of 5.0 g/L beef extract, 10.0 g/L high speed emulsifying machine (WL-500CY, Shanghai peptone and 5.0 g/L NaCl (pH 7.0). After the enrichment Wei Yu Mechanical and Electrical Manufacture Limited for 72 hr, 10 mL of the fermented broth was transferred Company, China). Span 80 and Tween 80 were purchased into 100 mL of modified mineral salts medium (MMSM) from Shanghai Shenyu Pharmaceutical and Chemical Lim- supplemented with paraffin or WFOs as carbon source. In ited Company in China. The fresh emulsion showed less this study, WFOs, a semi-solid mixture of vegetable oil and than 10% of emulsion breaking ratio at 35°C within 24 hr. lard, was obtained from the production process of a typical Cell suspension was prepared by suspending dried- Chinese fast food. The characteristics of this strain as well powder biodemulsifier in distilled water to achieve definite as the composition of MMSM were described elsewhere concentration. Before dosing, the samples were ultrasoni- (Liu et al., 2009). cally vibrated for 3 min to make the biodemulsifier fully Unless otherwise mentioned, liquid cultures (100 mL) dispersed. were incubated in flasks (250 mL) at 35°C on a rotary In the demulsification test, 2 mL of cell suspension was shaker (DKY-II, Shanghai Duke Automation Equipment added into a 20 mL graduated test tube containing 18 mL Company, China) at 140 r/min for 7 days. Initial pH values of the model emulsion. The test tubes were vigorously of the culture media was adjusted with 6 mol/L HCl and 2 inverted for 120 times to achieve complete mixing and then mol/L NaOH. left undisturbed in water baths at 35°C. The volume of 1.2 Methods of using waste frying oils as carbon source separated oil (on the top), separated water (on the bottom) and residual emulsion (in the middle) were recorded at To study the effect of pH on the synthesis of a biodemul- certain time intervals. Demulsification performance was sifier, the initial pH values of the culture media were evaluated by emulsion breaking ratio (R) according to the adjusted to 5, 6, 7, 8, 9, 10, and 11. Waste frying oils was following equations: added to MMSM at 4% (V/V) before sterilizing the culture V media. = − r × R (1 + ) 100% (1) To study the effect of mixed carbon sources on the Vo Va synthesis of biodemulsifier, waste frying oils and paraffin where, Vr is remaining emulsion volume, Vo is original at dosage ratios of 1%:3%, 2%:2% and 3%:1% (V/V) were emulsion volume, and Va is added cell suspension volume. applied together to the MMSM as mixed carbon sources to The blank test was conducted with 2 mL of distilled produce biodemulsifier. The initial pH was set at 7. water and it showed less than 10% of emulsion breaking To study waste frying oils as supplementary carbon ratio within 24 hr. source on the synthesis of biodemulsifier, strain S-XJ-1 1.5 Characterization was firstly inoculated on MMSM with a certain amount of paraffin as starting carbon source at the initial pH of 10. Fourier transform infrared spectroscopy (FT-IR) for the After a period of cultivation, a certain volume of sterilized samples was performed to give a preliminary character- waste frying oils was added to the fermented broth as ization of the main functional chemical groups present supplementary carbon source for the second fermentation on the biodemulsifier responsible for demulsification ca- process. The total dosage of carbon source was 4% (V/V), pability. Three dried-powder biodemulsifiers produced by with WFOs and paraffin at ratios of 1:3, 2:2 and 3:1. The tripalmitin, olein and tristearin were analyzed using FT- total fermentation cycle was 7 days. The adding time was IR (Nicolet 5700, Thermo, USA) adopting KBr disk set as day 1, 2, 3, 4, and 5 to compare the effect of adding technique. Equal weight of samples and equal weight of time on the production of biodemulsifiers. KBr were used to quantitatively compare the functional jesc.ac.cn 1022 Journal of Environmental Sciences 2011, 23(6) 1020–1026 / Jia Liu et al. Vol. 23 chemical groups. Biodemulsifier yield Demulsification 2 Results and discussion 5.0 100

2.1 Effect of initial pH on the synthesis of biodemulsifi- 4.0 80 er 3.0 60 In previous studies (Liu et al., 2011), it was found that biodemulsifier yield and its demulsifying ability was 2.0 40 improved by increasing initial pH when paraffin was used as the carbon source. The optimal pH was observed at

1.0 20 (%) ratio Demulsification 10 with the maximal biodemulsifier yield at 3.55 g/L and (g/L) yield Biodemulsifier highest demulsification ratio at 95.5% with the dosage of 1000 mg/L (data not shown). For using WFOs as carbon 0.0 0 source, it is necessary to study the effect of pH on the 1:3 2:2 3:1 WFO:Paraffin yield and demulsification capability of the biodemulsifier. Fig. 2 Biosynthesis of biodemulsifier at different mixed ratio of WFOs The results are shown in Fig. 1. It was found that this and paraffin. strain could not grow under the condition below pH 7 or above pH 9. The highest yield of biodemulsifier was was higher than that produced by WFOs as solely car- achieved at pH 8 with the value of 4.62 g/L. The biodemul- bon source at pH 7. It can be concluded that mixed sifier produced at pH 9 exhibited highest demulsification carbon sources is preferable for S-XJ-1 to synthesize the ratio at 84.5% with the dosage of 1000 mg/L. It was biodemulsifier, which is in accordance with other studies. found the yield of biodemulsifier was increased while Costa et al. (2009) found that yield of rhamnolipids and demulsification efficiency was decreased when paraffin polyhydroxyalkanoates was increased when mixed carbon was replaced by WFOs as carbon source, which indicated sources made of cassava wastewater and WFOs was used. that using WFOs as sole carbon source was not appropriate Deshpande et al. (1995) found animal and glucose to produce biodemulsifier. Considering using WFOs to could be used as compounded carbon source to synthesize partially replace the needed paraffin would decrease the sophorolipid. In another study, sophorolipid yield was production cost, effect of mixed carbon sources of paraffin increased by supplementing the animal fat to coconut and WFOs was investigated. residues (Felse et al., 2007). 2.2 Synthesis of biodemulsifier using waste frying oils However, demulsification capability of the biodemul- and paraffin as mixed carbon sources sifier produced by mixed carbon sources was decreased compared with that produced by paraffin or WFOs as sole The effect of WFOs and paraffin as mixed carbon carbon source. Highest demulsification ratio of 51% was sources on the biosynthesis of biodemulsifier was studied. achieved by the biodemulsifier produced by WFOs and As Fig. 2 shown, the biodemulsifier yield was increased paraffin at the ratio of 1%:3%, which indicated mixed as the ratio of WFO:paraffin increased. The maximal carbon source of WFOs and paraffin was not appropriate biodemulsifier yield (4.06 g/L) was achieved when the to produce efficiently demulsifying biodemulsifiers. The WFOs and paraffin was set at the ratio of 3%:1%, which reason of this result was not fully understood and should be further studied.

Biodemulsifier yield 2.3 Fed-batch fermentation of biodemulsifier by sup- plementing waste frying oils Demulsification 5.0 100 As discussed above, it is not effective using WFOs to produce biodemulsifiers by increasing initial pH or 4.0 80 combined with paraffin as mixed carbon sources. Hori et al. (2002) concluded that polyhydroxyalkanoate and 3.0 60 rhamnolipid production was enhanced when a sodium decanoate was supplemented at the second fermentation batch. Therefore, supplementing the WFOs at the second 2.0 40 fermentation batch was investigated. Figure 3a shows that the preferable order of ratios of

Demulsification ratio (%) ratio Demulsification Biodemulsifier yield (g/L) yield Biodemulsifier 1.0 20 WFOs and paraffin on the biodemulsifier yield was 2%:2% > 3%:1% > 1%:3%. The maximal yield was reached at 0.0 0 5.96 g/L when 2% of WFOs was supplemented to culture 7 8 9 medium cultured at day 4 with 2% of paraffin as starting Initial pH carbon source. Addition time of WFOs had little influence ff Fig. 1 Biosynthesis of biodemulsifier at di erent initial pH using waste on the demulsification capability of the biodemulsifier frying oils (WFOs) as carbon source. as shown in Fig. 3b. The preferable order of ratios of jesc.ac.cn No. 6 Application of waste frying oils in the biosynthesis of biodemulsifier by a demulsifying strain Alcaligenes sp. S-XJ-1 1023

WFO:Paraffin = 1%:3% WFO:Paraffin = 2%:2% WFO:Paraffin = 3%:1%

8.0 100 a b

7.0 90

6.0 80 5.0 70 4.0 Biodemulsifier yield (g/L) yield Biodemulsifier Demulsification ratio (%) ratio Demulsification

3.0 60 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Addition time of WFOs (day) Addition time of WFOs (day) Fig. 3 Biosynthesis of biodemulsifier at different supplementary time of WFOs. (a) biodemulsifier yield; (b) demulsification ability. two carbon sources on the demulsification capability was: 2.4 Mechanism of waste frying oils on the synthesis of 2%:2% < 3%:1% < 1%:3%, which was the reverse order biodemulsifiers of the influence on the yield of biodemulsifiers. Moreover, Tripalmitin, olein and tristearin were three main com- the demulsification ratio was higher than 80% for all the ponents of WFOs with the proportion of 25.3%, 32.9% biodemulsifiers produced through WFOs supplementary and 14.2%, respectively (Liu et al., 2009). To reveal the technology. Highest demulsification ratio was achieved at mechanism of WFOs utilization, these three main fatty 90% by the demulsifier which was produced by addition acid glycerides were separately used as sole carbon source of WFOs to paraffin at day 5 with a ratio of 1%:3%. at different initial pH. As shown in Fig. 4a, alkaline To decrease the carbon source cost and increase the condition had negative effect on the biodemulsifier yield biodemulsifier yield, the optimal fermentation condition for tripalmitin used as carbon source. Maximal yield of was set as: 3% (V/V) of WFOs was supplemented to the biodemulsifier was achieved at 3.4 g/L when tripalmitin fermented broth after cultured for 3 days with 1% (V/V) was used as carbon source with initial pH at 7. For olein paraffin as a starting carbon source. This result can be used as carbon source, highest yield of biodemulsifier explained as follows. As paraffin is composed of alkane reached 4.49 g/L at initial pH 9. The biodemulsifier yield while WFO is composed of fatty acid glyceride (Liu et was increased as initial pH increased from 7 to 9 while al., 2009), the latter is easier for S-XJ-1 to be utilized, it was decreased when pH was 10, which performed therefore, the biodemulsifier yield was higher. On the similar phenomenon with WFOs as carbon source (Fig. 1). contrary, alkane is more difficult to be utilized, therefore, However, the biodemulsifier produced by tristearin showed this strain have to regulate its cell surface to adapt to differently performance compared with the other two fatty the condition. In the adaptation process, the cell surface acid glycerides. The maximal yield of biodemulsifiers was of S-XJ-1 became more hydrophobic which increased its obtained at 1.54 g/L at pH 10, which was lower compared demulsifying ability. Based on these analysis, it can be with tripalmitin or olein as sole carbon source. From the drawn that S-XJ-1 firstly use paraffin as carbon source to above analysis, the preferable order for S-XJ-1 to utilize form hydrophobic cell surface during cultivation for initial the fatty acid glycerid was olein, tripalmitin, and tristerin, 3 days, and then more biodemulsifier was produced when which indicated that long-chain saturated fatty acid is WFOs was supplied.

Tripalmitin Olein Tristearin

5.0 100 a b

4.0 80

3.0 60

2.0 40

1.0 20 Biodemulsifier yield (g/L) yield Biodemulsifier Demulsification ratio (%) ratio Demulsification 0.0 0 7 8 9 10 7 8 9 10 Initial pH Initial pH Fig. 4 Effect of fatty acid glyceride on synthesis of biodemulsifier. (a) biodemulsifier yield; (b) demulsification ability. jesc.ac.cn 1024 Journal of Environmental Sciences 2011, 23(6) 1020–1026 / Jia Liu et al. Vol. 23 difficult for bacteria to utilize. The same conclusion was vibrations and N–H bending vibrations. Other significant drawn by Felse et al. (2007) on studying the synthesis peaks observed at 1450–1460 cm−1 correspond to C–H of rhamonolipid by Candida bombicola. Benincasa et bending vibrations and is common in compounds with al. (2002) found the preferable order of fatty acid was alkyl chains. olein, linolein, tripalmitin and tristerin for Pseudomonas However, there does exist two significant differences aeruginosa LBI to produce rhamonolipid, which was in among the three biodemulsifiers (Fig. 5). The adsorption accordance with this study. peak appearing 1737 cm−1 is related to the stretching As shown in Fig. 4b, initial pH had negative effect vibration of carbonyl group (C=O bond), which appear in on tripalmitin to synthesize biodemulsifiers but had little biodemulsifiers produced by tripalmitrin and olein other effect on the other two fatty acid glycerides. However, at than tristearin. Furthermore, the highest intensity of C=O pH 7, biodemulsifiers produced by tripalmitin, olein, and stretching vibration was achieved by the biodemulsifier tristearin showed highest demulsification ratios at 81%, produced by olein and rarely seen in that produced by 54%, and 90%, respectively. The preferable order for S-XJ- tristearin, which is corresponding to the performance of 1 to synthesize high efficient biodemulsifier was tristerin, demulsification capability of three biodemulsifiers. It is tripalmitin and olein, which was the reverse order for the predicted that the product of C=O group is occurred owing yield of biodemulsifier. It was indicated that saturated to the unsaturated bond in olein. The second difference long-chain fatty acid was effective for enhancement of existed in wavenumbers ranging from 3284 to 3294 cm−1 demulsification capability while unsaturated short-chain caused by C–H stretching vibrations and N–H stretching fatty acid was suitable for improvement of demulsifier vibrations, which indicated the nitrogen content. It was yield. found the biodemulsifier produced by tristearin showed Functional groups of three biodemulsifiers produced by more intensive vibration than the other two. It is predicted fatty acid glycerides were analyzed by FI-IR to reveal the that the nitrogen content of the biodemulsifier has great demulsifying impact factors. Biodemulsifiers produced at effect on its demulsification ratio. On the whole, it is esti- pH 7 were tested because the highest demulsification was mated that the decrease of C=O and increase of nitrogen achieved at this condition and the results are shown in content would contribute to higher demulsification ratio of Fig. 5. Three biodemulsifiers exhibited more or less similar a biodemulsifier. spectrum as follows. A broad absorbance with wavenum- In current studies on biological demulsifying mech- bers ranging approximately from 3500 to 3200 cm−1 with anism, it is predicted that demulsification capability is its maximal peak at 3300 cm−1 was seen. Absorbance in associated with hydrophobic and hydrophilic property of this region is caused by C–H stretching vibrations and N–H demulsifying bacteria. Stewart et al. (1983) found that stretching vibrations, which is a characteristic of carbon- Nocardia amarae could break the O/W emulsion owing to containing compounds with amino groups and signifies the its stronger hydrophobic cell surface. Demulsifying strain presence of intramolecular hydrogen bonding. Absorbance of Rhodococcus sp. PR-1 was also found hydrophobic peak is seen at 2923 cm−1 signifying the presence of with contact angle at 122.5◦ by Ma et al. (2006). Mean- C–CH3 bonding or long alkyl chains. The peak with while, other studies showed cell surface hydrophobicity highest absorbance in the spectrum was observed 1650– is relevant with cell surface compound such as mycolic 1660 cm−1, which signifies the presence of peptide group. acid, lipopolysaccharide and protein (Bunt et al., 1995). Adjacent to this peak was another high intensity peak It has been confirmed that cell surface hydrophobicity was at 1540–1550 cm−1, which is caused by C–H stretching increased with the increase in N/C of the cell surface (Al- Tahhan et al., 2000). From the above discussion, it can be predicted that demulsifying capability of bacteria is Tripalmitin associated with functional groups of the cell surface. In the authors’ opinion, biological demulsification is induced by integrated effects. Functional groups and element com- positions are crucial to its demulsifying capability. It is Olein suggested that key functional groups and element should be studied in detail to reveal the biological demulsification mechanism.

Transmittance 3 Conclusions

In this study, a high efficient demulsifying strain of Alcaligenes sp. S-XJ-1 was investigated on enhancement in synthesizing biodemulsifiers using waste frying oils. The Tristearin impact of waste frying oils as supplementary carbon source 4000 3500 3000 2500 2000 1500 1000 500 was proposed for the first time to produce biodemulsifi- Wavenumber (cm-1) er. To decrease the carbon source cost and increase the Fig. 5 FT-IR analysis of biodemulsifier produced by three fatty acid biodemulsifier yield, the optimal fermentation condition glycerides. was set as follows: 3% of waste frying oil was supplied jesc.ac.cn No. 6 Application of waste frying oils in the biosynthesis of biodemulsifier by a demulsifying strain Alcaligenes sp. S-XJ-1 1025 to the fermented broth after cultured for 3 days. Saturated as viable alternative sources for biosurfactant production. long-chain fatty acid was suitable for enhancement of World Journal of Microbiology and Biotechnology, 17(1): demulsification capability while unsaturated short-chain 61–69. fatty acid was preferable for demulsifier yield. The demul- Felse P A, Shah V, Chan J, Rao K J, Gross R A, 2007. sifying difference existed in the biodemulsifier produced Sophorolipid biosynthesis by Candida bombicola from by fatty acid glyceride indicted that the demulsification industrial fatty acid residues. Enzyme and Microbial Tech- nology, 40(2): 316–323. capability was associated with content of C=O and nitro- George S, Jayachandran K, 2009. Analysis of rhamnolipid gen element. In large pilot production of biodemulsifier, biosurfactants produced through submerged fermentation waste frying oils rich of long-chain saturated fatty acid using orange fruit peelings as sole carbon source. 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