Wastewater recycling technology for fermentation in polyunsaturated fatty acid production

Accepted Manuscript Wastewater recycling technology for fermentation in polyunsaturated fatty acid production Xiaojin Song, Zengxin Ma, Yanzhen Tan, Huidan Zhang, Qiu Cui PII: DOI: Reference:

S0960-8524(17)30304-8 http://dx.doi.org/10.1016/j.biortech.2017.03.034 BITE 17738

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

10 January 2017 28 February 2017 5 March 2017

Please cite this article as: Song, X., Ma, Z., Tan, Y., Zhang, H., Cui, Q., Wastewater recycling technology for fermentation in polyunsaturated fatty acid production, Bioresource Technology (2017), doi: http://dx.doi.org/ 10.1016/j.biortech.2017.03.034

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Wastewater recycling technology for fermentation in polyunsaturated fatty acid production Xiaojin Songa,c, Zengxin Maa,c,d, Yanzhen Tana, Huidan Zhanga,c,d, Qiu Cuia,b,c,* a Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, Shandong, China b Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, Shandong, China c Qingdao Engineering Laboratory of Single Cell Oil, Qingdao 266101, Shandong, China d University of Chinese Academy of Sciences, Beijing 100049, China

* Corresponding author: Qiu Cui Address: No. 189 Songling Road, Laoshan District, Qingdao, Shandong Province, P. R. China Tel: +86 532 80662706; Fax: +86 532 80662707 E-mail: [email protected]

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Abstract: To reduce fermentation-associated wastewater discharge and the cost of wastewater treatment, which further reduces the total cost of DHA and ARA production, this study first analyzed the composition of wastewater from Aurantiochytrium (DHA) and Mortierella alpina (ARA) fermentation, after which wastewater recycling technology for these fermentation processes was developed. No negative effects of DHA and ARA production were observed when the two fermentation wastewater methods were cross-recycled. DHA and ARA yields were significantly inhibited when the wastewater from the fermentation process was directly reused. In 5-L fed-batch fermentation experiments, using this cross-recycle technology, the DHA and ARA yields were 30.4 and 5.13 g L−1, respectively, with no significant changes (P > 0.05) compared to the control group, and the water consumption was reduced by half compared to the traditional process. Therefore, this technology has great potential in industrial fermentation for polyunsaturated fatty acid production. Keywords: Wastewater; Fermentation; Polyunsaturated fatty acid; Aurantiochytrium; Mortierella alpina

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1. Introduction

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Polyunsaturated fatty acids (PUFAs) have received worldwide attention because of their

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beneficial effects on human health (Kane & Correll, 2017; McGorry et al., 2017). In particular,

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docosahexaenoic acid (DHA, C22:6, n-3) and arachidonic acid (ARA, C20:4, n-6), are vital for

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infants to improve their visual and intelligence development, and also reduce risks associated with

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hypertension, inflammation, and certain cancers in adults (Doughman et al., 2013; Ramakrishnan

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et al., 2016; Zhang et al., 2017). Fish oil and animal viscera products are the major dietary sources

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of DHA and ARA (Graham et al., 2007; Sakuradani, 2010). However, because of emerging

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drawbacks such as limited fishery resources and the undesirable flavor of some animal viscera

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(Khozin-Goldberg et al., 2016), an increasing number of studies has been conducted to identify

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alternative sources of DHA and ARA. New alternative sources of DHA and ARA production

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include Aurantiochytrium sp. and Mortierella alpine, respectively (Lung et al., 2016; Li et al.,

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2015).

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Although nearly all aspects of microbial PUFA production have been extensively studied,

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such as strain screening (Cheng et al., 2016; Fu et al., 2016), medium optimization, and

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fermentation process control (Nie et al., 2013; Song et al., 2007), few studies have attempted to

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reduce the amount of wastewater generated in the production of microbial lipids or the reuse of

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supernatant wastewater. General industrial fermentation processes consume large amounts of

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water (Sun et al., 2013) and are accompanied by high biological oxygen demand (Coskun et al.,

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2012). Sewage treatment is not only expensive, but also requires large-capacity and high-power

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processing equipment (Guo et al., 2014; Ruiz-Rosa et al., 2016). Typically, the fermentation of

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Aurantiochytrium and Mortierella alpina for DHA and ARA production is carried out in the same

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factory in China, generating 100,000 tons of wastewater per year (Chen et al., 2016). Therefore, in

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fermentation, reducing fermentation wastewater discharge and reusing fermented wastewater are

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necessary and economical.

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The main limitation to recycling fermentative wastewater is the presence of cell growth

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inhibitors that accumulate during the fermentation process, including some components from the

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medium, metabolic processes, and secretions during cell growth (Muller et al., 2014). Although

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potential barriers to recovering fermentation wastewater have been widely discussed and a number

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of resolving methods have been proposed, few studies have been carried out. Chlorella

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pyrenoidosa was cultivated in wastewater from riboflavin fermentation to produce biomass and

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reduce the chemical oxygen demand (COD); the best volume ratio of wastewater was 5% and the

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treated wastewater could be recycled for fermentation after simple further treatment (Sun et al.,

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2013). Liu et al. used Lipomyces starkeyi to treat wastewater from monosodium glutamate

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fermentation and obtained biomass and lipid yields of 4.61 and 1.14 g L−1, respectively.

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Simultaneously, the COD decreased by 74.96%, reducing the cost of water treatment (Liu et al.,

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2012). Xu et al. utilized the wastewater from citric acid fermentation to produce methane, and

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then, the effluent was treated by air stripping and electrodialysis, and finally recycled as process

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water for citric acid fermentation. This process minimizes wastewater discharge from citric acid

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fermentation (Xu et al., 2016).

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To reduce wastewater discharge of fermentation and reduce the cost of wastewater treatment,

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which further reduces the total cost of DHA and ARA production, this study first analyzed the

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compositions of wastewater produced by Aurantiochytrium and M. alpina fermentation and then

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wastewater recycling technology for these DHA and ARA fermentation processes was developed.

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These results provide insight into the cost control strategy of PUFA production.

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2. Materials and methods

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2.1 Medium, strains, and culture conditions

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Aurantiochytrium sp. SD116 (CGMCC no. 6208) was reported in our previous study (Gao et

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al., 2013) and M. alpina SD003 (CGMCC no. 7960) was isolated by our laboratory and preserved

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in the China General Microbiological Culture Collection Center (CGMCC). An Erlenmeyer flask

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was used for culture and contained M1 medium for Aurantiochytrium (60 g L−1 glucose, 5 g L−1

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yeast extract, 5 g L−1 peptone, 5 g L−1 artificial seawater, pH 6.5) and GY medium for Mortierella

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(30 g L−1 glucose, 10 g L−1 yeast extract, pH 7.0). Both strains were cultured on a rotary shaker

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(200 rpm) at 25°C.

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For fed-batch fermentation, the initial fermentation medium for Aurantiochytrium contained

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100 g L−1 glucose, 20 g L−1 yeast extract, 10 g L−1 peptone, 7 g L−1 KH2PO4, 4 g L−1 (NH4)2SO4,

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and 2 g L−1 MgSO4 with an artificial sea salt concentration of 10 g L−1. The fermentation medium

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for Mortierella contained 30 g L−1 glucose, 20 g L−1 yeast extract, 4 g L−1 KH2PO4, 2 g L−1

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(NH4)2SO4, and 2 g L−1 MgSO4.

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2.2 Optimization of wastewater recycling conditions

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2.2.1 Water recycled from wastewater

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Different ratios of fresh water were replaced in fermentation medium. Different wastewater

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percentages (100%, 75%, 50%, 25%, and 12.5%) were used to replace the fresh water in the

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medium. The fermentation wastewater of Aurantiochytrium for DHA production was the

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supernatant obtained by centrifugation of the enzymatic hydrolysate of Aurantiochytrium, which

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was the product of papain enzymolysis after fermentation with the initial fermentation medium.

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The fermentation wastewater of Mortierella for ARA production contained the supernatant

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obtained by filtering the zymotic fluid following fermentation and fermentation medium using a

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filter-press.

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2.2.2 Exchange wastewater recycled using wastewater from PUFA fermentation

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In this experiment, to investigate the effects of one type of wastewater on PUFA fermentation,

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fresh water in the fermentation medium was replaced with exchanged wastewater at ratios of 20%,

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40%, 60%, 80%, and 100%.

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2.2.3 Fed-batch fermentation experiments for DHA and ARA production using exchange

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wastewater recycling technology

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Fed-batch fermentation experiments were performed in 5-L Biostat® B plus bioreactors

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(Sartorius Lab Products, Göttingen, Germany), which were equipped with controllers for pH,

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temperature, agitation, and dissolved oxygen concentration (DO). The initial medium volume of

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fed-batch cultures was 2.5 L and temperature was maintained at 25°C. The agitation speed

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automatically varied from 300 to 1000 rpm at a fixed air flow rate of 1.3 vvm to maintain the DO

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at 20% air saturation. The pH was maintained by adding 2 M NaOH or 2 M HCl. To control foam

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formation, 0.6 mL antifoam was added at the beginning of the run. Intermittent glucose feeding

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was supplied to maintain the residual glucose concentration at approximately 5–15 g L−1 by

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feeding with a 60% (W:V) glucose stock solution.

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2.3 Biomass determination

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The biomass of Aurantiochytrium and Mortierella were expressed as dry cell weight.

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Ten-milliliter samples were centrifuged at 7000 ×g and 4°C for 10 min and then freeze-dried to a

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constant weight at −50°C for approximately 60 h.

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2.4 Analytical methods

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Ion concentrations in the wastewater were analyzed by ion chromatography (IC, CIC-D160,

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SHINE, Qingdao, China) equipped with SH-AC-4 column for anions (Cl−, SO42−, NO3−, PO43−)

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and SH-CC-3 column for cations (Na+, K+, NH4+, Mg2+) (SHINE) as described by Zhang et al.

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(2017). Residual glucose in the fermentation medium was analyzed by a biosensor equipped with

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a glucose oxidase electrode (SBA-40E, Institute of Biology, Shandong Academy of Sciences,

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Shandong, China). High-performance liquid chromatography (RIGOL L-3000; Beijing, China)

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was used to measure the levels of free amino acids (Khan et al., 1998). The mobile phase used for

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isocratic elution contained methanol and 50 mM sodium phosphate buffer (pH 6.5) in a ratio of

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3:97 (v/v). Before use, the mobile phase was filtered through a 0.22-μm membrane filter and

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degassed by ultrasonication. The flow-rate was 1.0 mL min−1 and column temperature was 40°C.

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Compounds were monitored at 215 nm with a UV absorbance detector using a LiChrospher 100

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RP-18 (250 × 4 mm I.D., EMD Millipore, Darmstadt, Germany) column with a 5-mm particle size.

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An Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) and refractive index detector were

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used to analyze compounds, including cellobiose, xylose, acetate, and lactate (Mohr et al., 2013).

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Additionally, 5 mM H2SO4 was used as the mobile phase at 55°C at a flow rate of 0.5 mL min−1.

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2.5 Lipid extraction and fatty acid analysis

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Total lipids were extracted using a combination of chloroform and methanol (2:1, v/v) as

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previously described (Ma et al., 2015). The extracted lipids were weighed and dissolved in

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chloroform. Next, fatty acid methyl esters (FAMEs) were obtained by incubating the lipids at

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85°C for 150 min in the presence of 2% (v/v) sulfuric acid in methanol. The FAMEs were then

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extracted using hexane and evaluated by gas chromatography. An HP-INNOWAX (30 m × 0.25

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mm × 0.25 μm; Agilent, Technologies, Santa Clara, CA, USA) capillary column was used to

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separate the FAMEs. The oven temperature was initially set to 100°C for 1 min, and then

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increased to 250°C at a rate of 15°C min−1 and then maintained at 250°C for 5 min. The split ratio

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was 1:19 and nitrogen was used as the carrier gas. Peak detection was performed using a flame

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ionization detector. The temperature of the injection port and flame ionization port was 260°C and

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injection volume was 1 μL (Song et al., 2013).

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2.6 Calculation and statistical analysis

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All results are reported as the mean ± standard error of the mean and significant differences

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(P < 0.05, P < 0.01) between means were compared using the t-tests with SPSS v13.0 software

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(SPSS, Inc., Chicago, IL, USA).

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3 Results and discussion

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3.1 Nutrient concentrations in wastewater from PUFA fermentation

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The final volume of Aurantiochytrium fermentation broth for DHA production was 1.6-fold

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higher than the initial volume because medium was added during the fermentation process, while

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the volume of Mortierella fermentation broth for ARA production did not change because of the

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balance between feeding and evaporation. Compounds (including cations, anions, carbohydrate,

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organic acids, and amino acids) in the wastewater following DHA and ARA production were

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analyzed by immunocytochemistry and high-performance liquid chromatography. Five cations

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(Na+, K+, NH4+, Mg2+, and Ca2+) and four anions (Cl−, NO3−, SO42−, and PO43−) were detected. As

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shown in Table 1, after fermentation, the concentrations of NH4+, SO42−, and PO43− were

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significantly decreased because these ions were absorbed and utilized by microorganisms for

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biomass production (Wang et al., 2016), while the Mg2+ concentration was reduced because it was

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used as a coenzyme. The concentrations of other ions, such as Na+, K+, Ca2+, and Cl−, showed

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nearly no change from the levels in the initial medium, as they maintained the osmotic pressure or

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functioned as carrier assistants for substrate transport (Hu et al., 2015).

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The organic substances detected in wastewater, including carbohydrates, organic acids, and

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amino acids, are shown in Table 2. Apart from glucose, trace amounts of xylose and cellobiose

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(0.005 and 0.013 g L−1, respectively) were detected in wastewater from DHA production, which

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may have been produced through enzymolysis of Aurantiochytrium biomass. Lactic acid was the

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major organic acid detected in Aurantiochytrium fermentation broth at a concentration of 0.07 g

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L−1. Because of the enzymolysis of Aurantiochytrium biomass, large amounts of amino acids were

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dissolved in the wastewater of DHA fermentation, and the final concentration of total amino acids

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was approximately 5 g L−1. In contrast, only 0.243 g L−1 residual glucose was detected in

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wastewater from ARA production, while 0.412 g L−1 acetate was found in the same fermentation

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broth. Compared to Aurantiochytrium fermentation, the final concentration of residual total amino

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acids was 0.57 g L−1 in the fermentation broth of Mortierella.

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3.2 Effects of recycled wastewater from fermentation on biomass and PUFA production with

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Aurantiochytrium and M. alpine

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A series of experiments were carried out to detect the effect of reusing wastewater at different

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ratios (12.5%, 25%, 50%, 75%, and 100%) on biomass and PUFA (DHA and ARA) production.

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As shown in Figure 1A, Aurantiochytrium fermentation was significantly inhibited (P < 0.01)

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when the wastewater recovery ratio was greater than 25%. In the treatment groups with a 75% and

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100% replacement ratio, the final biomasses were only 11.8 and 4.9 g L−1, while DHA production

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was 1.94 and 0.74 g L−1, respectively. Biomass and DHA production in the control group were

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23.7 and 4.79 g L−1, respectively.

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In general, organic acids and alcohols (such as formic acid, acetic acid, ethanol, etc.)

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produced by cell metabolism can inhibit cell growth (Pereira et al., 2016; Semchyshyn et al.,

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2011). However, based on the results of component analysis of wastewater from the fermentation

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and hydrolysate of Aurantiochytrium, the concentrations of the organic acids acetic acid and lactic

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acid were only 0.005 and 0.07 g L−1, respectively, indicating that these acids were not the main

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cause of growth inhibition for Aurantiochytrium. Some studies showed that specific signaling

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molecules such as amino acids, free fatty acids, small peptides, and small RNA produced by cell

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secretion or apoptosis during fermentation can also cause severe growth inhibition (Bosma et al.,

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2008; Fu et al., 2014; Mercade et al., 2003). Mazzoleni et al. (2015) demonstrated that the

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accumulation of self-produced inhibitory compounds of yeast during continuous and batch

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fermentation were the major causes of growth inhibition of Saccharomyces cerevisiae. When the

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concentration of the inhibitor accumulated to a certain threshold, growth inhibition was observed,

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resulting in growth inhibition during yeast fermentation (Mazzoleni et al., 2015). The results of

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this study were similar to those of Mazzoleni et al.; therefore, the high concentrations of

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self-produced inhibitors may have inhibited the growth of Aurantiohytrium when the wastewater

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was directly reused as a medium water for DHA fermentation.

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Different results were obtained for Mortierella alpine fermentation; no significant changes or

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only slight changes were observed in biomass and ARA production (Figure 1B). The lowest

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biomass and ARA yield in the treatment group were 19.7 and 3.01 g L−1; these values were 22.6

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and 3.44 g L−1, respectively, in the control group. Therefore, successive multi-cycle experiments

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were carried out to investigate the effects of cycle times on biomass and ARA yields (Figure 1C).

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In the second recycle, M. alpina growth was inhibited by approximately 42.7% (only 12.2 g L−1).

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The results of the third and fourth rounds showed even lower yields, with biomass reduced by

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61.9% and 74.6%, respectively.

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As shown in Table 2, a high acetic acid concentration may be the main cause of growth

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inhibition of M. alpina during multi-cycle fermentation. When the concentration of acetic acid in

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the fermentation broth was greater than 2–4 g L−1, cell growth was inhibited (Ding et al., 2013;

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Zeng et al., 2013). When acetic acid stress occurs, intracellular ATP levels are significantly

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reduced, slowing ATP-dependent proton symport and inhibiting transcription of nutrient

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transporters, resulting in decreased nutrient uptake (Ding et al., 2013). In this study, the

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concentration of acetic acid in the fermentation broth of M. alpina was 0.412 g L−1, and further

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accumulated over longer fermentation times; thus, the much higher concentration of acetic acid

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inhibited growth during ARA fermentation.

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Thus, these two fermentation processes cannot be conducted when their own fermentation

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wastewater is used, although the first re-use of wastewater has little effect on the ARA

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fermentation process.

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3.3 Effects of exchanged recycled wastewater on DHA and ARA production

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The effects of exchanged recycled wastewater on DHA and ARA fermentation were

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evaluated. As shown in Figure 2A, when the wastewater of ARA fermentation was reused rather

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than fresh water in the medium of DHA fermentation, none of the treatment groups (including the

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five replacement ratios of 20%, 40%, 60%, 80%, and 100%) showed significant changes in

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biomass and DHA yield (P > 0.05). DHA production was slightly decreased by 5.7% when 100%

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freshwater was replaced. Additionally, the biomass and ARA yield of M. alpina showed no

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significant differences from the control group (P > 0.05), although ARA yield was 3.3 g L−1, which

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was slightly higher than the control value. Both the variation range of biomass and ARA yield

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were less than 5% (Figure 2B) when the replacement ratio was up to 80% (replacement rate 80%,

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60%, 40%, and 20%). In contrast, M. alpina growth was significantly inhibited (P < 0.05) when all

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freshwater was replaced with wastewater from DHA fermentation (replacement rate 100%

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treatment group). The biomass and ARA yield were 18.2 and 2.41 g L−1, respectively, showing

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15.7% and 26.2% decreases compared to the control group.

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Aurantiochytrium was previously shown to efficiently uptake acetic acid as a carbon source

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for growth (Song et al., 2013). Therefore, 0.412 g L−1 acetic acid in ARA fermentation wastewater

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will not affect the growth of Aurantiochytrium. Additionally, signaling molecules that may

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specifically inhibit the growth of Aurantiochytrium in DHA fermentation wastewater will not

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inhibit the growth of M. alpina and may be taken up as substrates by M. alpina. However, because

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of the low halophilic nature of M. alpina (Nisha et al., 2011), an excessive salt concentration

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would inhibit its growth when fermentation water was completely replaced with wastewater from

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DHA fermentation.

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Table 3 and 4 show the fatty acids compositions of Aurantiochytrium and M. alpina. The

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types and percentages of fatty acids changed insignificantly under exchanged recycled wastewater

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conditions. DHA and ARA remained as the highest proportions of fatty acids; the differences from

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control group were less than 5.3% and 9.4%, respectively. These results are similar to those for

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microalgae cultured in industrial wastewater (Kamyab et al., 2016), and thus the effects of

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wastewater recycling on PUFAs production mainly resulted from the inhibition of cell growth and

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oil accumulation rather than changes in the fatty acid composition.

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3.4 Effects of fed-batch fermentation on DHA and ARA production using exchange wastewater

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recycled technology

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The 5-L fed-batch fermentation experiments were carried out to verify the results of the

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shake flask experiments. The freshwater in the DHA fermentation medium was completely

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replaced with wastewater form ARA fermentation. For the first and second cycles, DHA yields

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were 31.7 and 30.4 g L−1, while in the control group (freshwater added), DHA yield was 30.0 g

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L−1. After the third cycle, DHA yield was 28.7 g L−1, which was slightly decreased (4.5%) but not

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significantly changed (P > 0.05) compared with the control group (Figure 3A). According the

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results of the shake flask experiments, freshwater in the ARA fermentation medium was replaced

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80% with wastewater from DHA fermentation. As shown in Figure 3B, after the second cycle,

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ARA yield was reduced by 3.7% compared to the control group (5.13 vs. 5.33 g L−1). However,

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ARA yield significantly decreased (P < 0.01) to 4.03 g L−1 (approximately -24.4%) after the third

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cycle. Based on the results of the fed batch fermentation experiments, the new wastewater reuse

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process can be used at least twice with no decreases in DHA and ARA yields.

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Based on the above results, water recycle technology for the DHA and ARA fermentation

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process is shown in Figure 4A. Compared with the traditional fermentation process (Figure 4B),

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freshwater consumption decreased production by 46.2% after two cycles, and this percentage was

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increased to 51.9% when the wastewater was reused three times (calculation method is shown in

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Supplemental data table S1).

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4 Conclusion

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In summary, a wastewater cross-recycle technology for DHA and ARA fermentation was

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developed in this study. Using this technology, water consumption and discharge can be reduced

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by half, which can greatly reduce costs. Therefore, this technology has great potential for

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large-scale or industrial fermentation for PUFA production.

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Acknowledgments

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This work was supported by the National Key Research and Development Program (no.

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2016YFA0601400), National Natural Science Foundation of China (no. 41306132), and National

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High Technology Research and Development Program of China (no. 2014AA021701). This study

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is a contribution to the international IMBER project.

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377 378 379 380 381

17

382

Figure Captions

383

Figure 1 Effects on cell growth, lipid accumulation, and PUFA (DHA & ARA) production by

384

different freshwater replacement ratios using fermentation wastewater. A, Effects on cell growth,

385

lipid accumulation, and DHA production using different replacement ratio; B, effects on ARA

386

production using different replacement ratios; C, effects on ARA production for different recycling

387

times; **, P < 0.01.

388

Figure 2 Effects on cell growth, lipid accumulation, and PUFA (DHA & ARA) production by

389

different freshwater replacement ratios using exchanged wastewater. A, Effects on DHA

390

production; B, effects on ARA production; *, P < 0.05.

391

Figure 3 Effect of exchanged wastewater recycling technology on cell growth, lipid accumulation,

392

and PUFA (DHA & ARA) production in fed-batch fermentation. A, Cell growth, lipid

393

accumulation, and DHA yield in exchanged wastewater recycle fermentation; B, cell growth, lipid

394

accumulation, and ARA yield in exchanged wastewater recycle fermentation; **, P < 0.01.

395

Figure 4 Innovation and traditional process control flow chart. A, New wastewater cross-recycle

396

technology for DHA and ARA fermentation developed in this study; B, traditional fermentation

397

process for DHA and ARA fermentation.

398

18

399 400

Tables Table 1 Determination of ion concentration in fermentation wastewater

Concentration (M) AU

MA

0.11235

nd

NH4

0.02378

0.01058

K+

0.00622

0.00477

Mg

0.00194

0.00015

Ca2+

0.00033

0.0001

0.11242

nd

Cations Na+ +

2+

Anions Cl− NO3−

0.00006

nd

SO4

2−

0.00643

0.00528

PO43−

0.00097

0.00174

401

AU: wastewater from Aurantiochytrium fermentation for DHA production

402

MA: wastewater from Mortierella alpina fermentation for ARA production

403

nd: not detected

404

19

405

Table 2 Analysis of organic substances in wastewater

Concentration (g L−1) AU

MA

Glucose

1.325

0.243

Xylose Cellobiose

0.005 0.013

nd nd

Acetate

0.005

0.412

Lactate

0.07

nd

Glu

0.0176

0.0237

Arg

2.0628

0.0788

Lys

1.2396

0.0876

Ala

0.3152

0.0352

Thr

0.0092

0.0075

Gly

0.028

0.019

Val

0.006

0.016

Ser Ile Met His Asp Cys Trp

0.0096 0.03 0.0124 0.03 1.1312 0.0148 0.0216

0.0044 0.0124 0.0214 0.0815 0.1272 0.054 0.0016

Amino acids

406

AU: wastewater from Aurantiochytrium fermentation for DHA production

407

MA: wastewater from Mortierella alpina fermentation for ARA production

408

nd: not detected

409

20

410

Table 3 Comparison of fatty acid compositions of Aurantiochytrium after culture with exchanged

411

wastewater in different replacement ratios

Fatty acid C14:0 C15:0 C16:0 C16:1 C17:0 C18:0 C20:0 C20:4 C20:5 C22:5 C22:6 (DHA) 412

CK 1.83 6.86 25.75 0.23 1.53 1.92 1.26 0.87 0.55 11.95 47.14

20% 2.36 7.26 25.36 0.35 1.19 1.83 0.97 0.95 0.47 12.35 46.73

Content (% total fatty acids) 40% 60% 1.95 1.38 6.35 7.42 26.14 25.33 0.36 0.16 1.54 1.08 1.65 2.05 1.06 1.11 0.91 0.79 0.59 0.61 9.72 12.01 49.64 47.78

80% 1.95 6.91 24.79 0.21 1.33 2.01 1.03 0.91 0.59 11.65 48.44

100% 1.59 6.67 25.43 0.19 1.25 1.79 0.93 1.06 0.64 13.37 46.89

CK: control group

413

21

414

Table 4 Comparison of fatty acid compositions of Mortierella alpina after culture with exchanged

415

wastewater in different replacement ratios

Fatty acid C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 C20:3 C20:4 (ARA) 416

CK 1.46 9.72 17.63 16.21 5.88 3.16 2.15 43.31

20% 1.05 11.51 19.91 14.05 5.39 4.01 2.76 40.76

Content (% total fatty acids) 40% 60% 2.03 1.95 10.35 10.81 21.17 21.5 11.32 12.35 4.95 5.19 3.27 3.93 3.13 2.21 43.59 41.55

80% 1.87 8.16 21.74 14.72 4.26 3.77 1.54 43.82

100% 1.82 9.58 18.69 16.98 5.74 4.32 2.86 39.24

CK: control group

417

22

418

Figures

419

Figure 1

420

421

422 423 23

424

Figure 2

425

426 427

24

428

Figure 3

429

430 431

25

432

Figure 4

433

434 435

26

436 437

HighLights

438 439 440 441 442 443

 A wastewater cross-recycle technology for DHA and ARA fermentation was developed  The new technology has no negative effects on DHA and ARA yields  The DHA and ARA yields were 30.4 g L-1 and 5.13 g L-1 respective after two cycles  Water consumption and discharge would reduce by half using this technology

444

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445

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