Microb Ecol (2014) 67:454–464 DOI 10.1007/s00248-013-0332-y

PHYSIOLOGY AND BIOTECHNOLOGY

Screening of Marine Bacterial Producers of Polyunsaturated Fatty Acids and Optimisation of Production Ahmed Abd El Razak & Alan C. Ward & Jarka Glassey

Received: 3 May 2013 / Accepted: 12 November 2013 / Published online: 30 November 2013 # Springer Science+Business Media New York 2013

Abstract Water samples from three different environments including Mid Atlantic Ridge, Red Sea and Mediterranean Sea were screened in order to isolate new polyunsaturated fatty acids (PUFAs) bacterial producers especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Two hundred and fifty-one isolates were screened for PUFA production and among them the highest number of producers was isolated from the Mid-Atlantic Ridge followed by the Red Sea while no producers were found in the Mediterranean Sea samples. The screening strategy included a simple colourimetric method followed by a confirmation via GC/MS. Among the tested producers, an isolate named 66 was found to be a potentially high PUFA producer producing relatively high levels of EPA in particular. A Plackett–Burman statistical design of experiments was applied to screen a wide number of media components identifying glycerol and whey as components of a production medium. The potential low-cost production medium was optimised by applying a response surface methodology to obtain the highest productivity converting industrial by-products into value-added products. The maximum achieved productivity of EPA was 20 mg/g, 45 mg/l, representing 11 % of the total fatty acids, which is approximately five times more than the amount produced prior to optimisation. The production medium composition was 10.79 g/l whey and 6.87 g/l glycerol. To A. A. El Razak : J. Glassey (*) School of Chemical Engineering and Advanced Materials, CEAM, Merz Court, Newcastle University, Newcastle upon Tyne NE1 7RU, UK e-mail: [email protected] A. A. El Razak Botany Department, Faculty of Science, Mansoura University, Mansoura, Egypt A. C. Ward School of Biology, Newcastle University, Newcastle, UK

our knowledge, this is the first investigation of potential bacteria PUFA producers from Mediterranean and Red Seas providing an evaluation of a colourimetric screening method as means of rapid screening of a large number of isolates.

Introduction Health Benefits of Eicosapentaenoic Acid Eicosapentaenoic acid (EPA) is an omega-3 member of longchain fatty acids with 20 carbon atoms and five nonconjugated cis double bonds. EPA is an essential part of glycolipids and phospholipids, necessary components of plasma membranes, acting as precursors for many hormones and hormone-like regulatory molecules, such as eicosanoids. In addition, they demonstrate marked effect on human health [1]. The health benefits of EPA include the lowering of plasma cholesterol and decreasing the incidence of breast, colon and pancreatic cancers [2]. The ability of EPA to prevent and cure most of the blood-circulatory diseases indicates that EPA has an anti-aggregatory character and is essential in maintaining homeostasis [3]. Omega-3 fatty acids, especially EPA, were reported to have a protective role as antioxidants [4]. The antioxidant effect of EPA is thought to be due to the formation of a barrier preventing the diffusion of the oxygen reactive molecules across the membranes rather than the activation of catalase activity [5]. Microbial Eicosapentaenoic Acid Fish, such as mackerel, sardines and herring, are basic sources for the commercial production of omega-3 fatty acids, including EPA [6]. In addition to the typical fishy smell and taste, fish oil as the main source of EPA in the market is not expected

Screening of Marine Bacterial Producers of Polyunsaturated Acids

to meet the ever-growing global demand due to the overfishing problem [7]. Another concern regarding the use of fish oil is the environmental pollution of marine ecosystems especially with heavy metals and dioxins which accumulate in fish represents a hazard to human health [8]. Also, both the composition and quantity of polyunsaturated fatty acids (PUFAs) in fish rely on the species, season and geographical location of the capture. In addition, the refining conditions can affect the quality of PUFAs [9]. Fish oil contains a complex mixture of fatty acids with antagonistic effect which may inhibit cancer chemotherapy process [10]. As a result, scientists are trying to find alternative ways of obtaining PUFAs in order to satisfy the increasing demand for omega-3 products, and microbial sources were identified as the most attractive potential sources [11]. In addition, bacterial oil rich in omega-3 was recently proven to have a competitive stability compared to fish oil [12]. Microbial oils, also called single cell oils (SCOs), have been manufactured on an industrial scale since the 1980s. Microorganisms tend to produce SCOs under a certain set of conditions and divert the metabolism towards lipid formation and accumulation. Bacterial EPA accumulates only within the cellular membrane phospholipids mainly in the form of phosphatidyl ethanolamine [13]. The phospholipid nature of bacterial EPA could be advantageous over the triglyceride form in fungi and algae as EPA is an orthomolecule whose target is solely cell membranes [14, 15].

Natural Habitat of Microbial Eicosapentaenoic Acid Producers EPA-producing bacteria identified to date belong mainly to Shewanella , Photobacterium , Colwellia , Vibrio and Psychromonas [16, 17]. Generally, most of these EPA producers are psychrophilic and piezophilic and originate from the polar regions and the deep sea [18]. The high EPA percentage in such microorganisms plays a critical role in their adaptation to these extreme environments, where the high PUFA percentage gives the plasma membrane the ability to remain fluid under low temperatures [19]. EPA was also found to play a critical role in the bacterial membrane organisation and cell division especially at low temperature [20]. EPA protects the bacterial cells not only against the extreme cold environments but also against the high pressure stress, which explains the abundance of EPA among the psychrophilic and piezophilic bacteria [21]. Although most of the known microbial EPA producers were isolated from the deep sea and cold environments, some mesophilic and shallow water bacteria were found to produce EPA. Two Shewanella strains (ACEM 6 and ACEM 9), isolated from a temperate, humus-rich river estuary in

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Tasmania Australia, were able to produce a high level of PUFAs at relatively high incubation temperatures (10.2 % at 24 °C) [22]. The main target of this work is to screen different and nontraditional environments for potential new EPA high producers to evaluate a novel screening method and to enhance the productivity of the chosen isolate(s) by applying a statistical design of experiment.

Materials and Methods Sample Collection and Cultivation Conditions Deep sea core sediments and water samples were collected from three different areas as follows: Mediterranean Sea, Red Sea and Mid-Atlantic Ridge. Mid-Atlantic Ridge samples were deep sea core sediment and fluff samples collected from the Mid-Atlantic ridge by research personnel at the Dove Marine laboratory, Newcastle University and kindly provided for this research, while the Red and Mediterranean Sea samples were collected in Egypt by a diver at 25-m depth (latitude and longitude of 27.24–33.89 for the Red Sea sampling area and 31.20–29.91 for the Mediterranean Sea sampling area). These samples were diluted in sterile water by serial dilution to 10−7. One hundred microliters from the last three dilutions were plated out on marine agar plates and incubated at 20 °C for 48 h. Colonies were collected depending on the morphological variations among them, numbered and cultured on slants for further work. Colourimetric Screening for EPA Producers After 24 h of growth in artificial sea water (ASW) medium at 20 °C in an orbital shaker incubator at 160 rpm, 0.1 % w/v of the dye 2,3,5-triphenyltetrazolium chloride (TTC) was added to the growth broth. Tubes were incubated at 20 °C for 1 h. The formation of red colour was considered to be a positive result [23]. Fatty Acid Methyl Ester Preparation Twenty milligrams of freeze-dried cells were suspended in 2 ml of 5 % methanolic HCl and heated at 70 °C in a water bath for 2 h in sealed glass tubes. The tubes were cooled at room temperature for 30 min, then 1 ml distilled water was added and the tubes were vortexed. To extract the fatty acid methyl esters (FAME), 2-ml hexane was added and vigorously vortexed. The tubes were kept till two layers were formed. The upper layer was transferred into a clean tube and dried under nitrogen [24]. EPAwas extracted from whole intact cells in order to avoid oxidation resulting from cell disruption.

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Gas Chromatography/Mass Spectroscopy

Table 1 Variables investigated for EPA production via Plackett–Burman design for isolate 66

Gas chromatography/mass spectroscopy (GC/MS) analysis was performed on an Agilent 7890A GC in split mode injector at 280 °C linked to Agilent 5975C MSD with the electron voltage 70 eV, source temperature 230 °C, quad temperature 150 °C, multiplier voltage 1,800 V and interface temperature 310 °C, controlled by a HP Compaq computer using ChemStation software. The sample (1 μl) in hexane was injected using HP7683B auto sampler with the split open. After the main solvent peak had passed the GC temperature programme and data acquisition commenced. Separation was performed on an Agilent-fused silica capillary column (30 m× 0.25 mm i.d) coated with 0.25 μm dimethyl poly-siloxane (HP-5) phase. The GC was temperature programmed from 30 to 130 °C at 5 °C/min then to 300 °C at 20 °C/min and held at a final temperature for 5 min with helium as the carrier gas (flow rate of 1 ml/min, initial pressure of 50 kPa, split at 10 ml/min). Peaks were identified and labelled after comparison of their retention time and mass spectra with those of the NIST05 library using >90 % fit.

Variables

Code Unit Minimum level (−) Maximum level (+)

Yeast extract Glycerol Whey Meat peptone

X1 X2 X3 X4

g/l g/l g/l g/l

0 0 0 0

1 1 1 1

Maltose Mannitol Urea Na2HPO4 Glutamic acid Glycine Fish peptone Hy-Soy Sodium acetate Casein MgSO4 NaCl Lactose Dummy

X5 X6 X7 X8 X9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18

g/l g/l g/l g/l g/l g/l g/l g/l g/l g/l g/l g/l g/l –

0 0 0 0 0 0 0 0 0 0 0 10 0 Distilled water

1 1 1 0.89 1 1 1 1 1 1 5 30 1 Bi-distilled water

Growth in Artificial Sea Water A loopful of biomass from culture plates was transferred into 250-ml flask containing 50 ml of ASW medium (peptone 3.5 g/l; yeast extract 3.5 g/l; NaCl 23 g/l; MgCl2 5.08 g/l; MgSO4 6.16 g/l; Fe2 (SO4)3 0.03 g/l; CaCl2 1.47 g/l; KCl 0.75 g/l; Na2HPO4 0.89 g/l; NH4Cl 5.0 g/l) [25] and allowed to grow at 20 °C in an orbital shaker incubator at 160 rpm for 24 h. The effect of different temperatures, pH and NaCl was tested on all PUFA producers by cultivating them on ASW medium. Growth in Production Medium The growth was performed in 250-ml sterile flasks with 50 ml of given media at 15 °C for 2 days in an orbital shaker incubator at 160 rpm. Final biomass from each flask was collected into a 50-ml Falcon centrifuge tube and centrifuged at 6,000 rpm for 15 min. The cell pellets were transferred into a 1.5-ml screw tube and freeze-dried overnight. Initial Screening by Plackett–Burman Design Each variable was investigated at a high (+) and a low (−) level. The variables were tested on the presence/absence principle, where the low level was always zero except for sodium chloride as the marine isolates tested were unable to grow in the complete absence of it. In addition to the 17 tested variables, a dummy variable was used to evaluate the standard error of the experiments as shown in Table 1.

The difference between the minimum and maximum levels tested should be neither small, as it may not show the effect, nor large, as it could mask the effect of the others [26]. This screening experiment allowed an objective comparison of the significance and main effect of each factor on the amount of EPA produced by the isolate under investigation. The main effects of each variable were calculated using Eq. 1.   − 2 ∑Y þ i −∑Y i E ðXiÞ ¼ ð1Þ N Where E (Xi) is the effect of the tested variable and Y i+ and are the responses at maximum and minimum levels, respectively. The significance level (P value) of each variable was determined using Student's t test (Eq. 2). Y i−

t ðXiÞ ¼

E ðXiÞ SE

ð2Þ

Where (SE), the standard error of variables, is calculated as the square root of the variance of an effect. Any variable with P F Sum of squares F value p value prob > F A-whey B-glycerol AB A2 B2

30.43 5.09 29.80 94.95 85.80

3.06 0.51 3.00 9.55 8.63

0.1237 0.4975 0.1270 0.0176 0.0218

2,159.90 303.49 130.93 7.54 128.47

29.27 4.11 1.77 0.10 1.74

from the GC experiments which require the cultivation and FAME extraction of the isolate under the investigation. During this research, 65 samples resulted in a positive TTC

A 10

0.1443 0.6483 0.5116 0.0105 0.0212

EPA conc. (mg/l)

15.00

5

11.50

B: Glycerol (g/l)

(g/l)

10.25

B: Glycerol

2.70 0.23 0.48 12.00 8.75

response. The total fatty acids from all these isolates were extracted and examined by GC/MS (section Gas Chromatography/Mass Spectroscopy), showing that only 14

B

EPA yield (mg/g)

13.00

9.66 0.81 1.71 42.96 31.31

0.0010 0.0821 0.2245 0.7585 0.2285

7.50

44.6431 40

8.00 30

4.50

4.75 5

51.3889

20

10

13.8209

0 0

2.00 1.50

1.00 4.87

8.25

11.63

15.00

1.00

4.50

8.00

A: Whey (g/l)

11.50

15.00

A: Whey (g/l)

C

EPA %

15.00

2 4

4

6

B: Glycerol (g/l)

11.75

8.50

10 5.25

9.07646 4 2.00 2.00

6 8 5.25

8.50

11.75

15.00

A: Whey (g/l)

Fig. 2 Colour contour plot showing the optimal interaction between the variables for maximum EPA production: a EPA yield (mg/g), b EPA concentration (mg/l) and c EPA percentage of the total fatty acids

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isolates were able to produce PUFAs. This indicates a relatively high false-positive rate of this method of 79 %. However, none of the negative TTC isolates tested showed any sign of the ability to produce PUFAs giving 0 % false-negative rate. The main disadvantage of using this screening method is the high false-positive rate with not all the positive TTC isolates being PUFA producers. Another problem in applying the TTC method is the sensitivity of the result to the incubation time. If the incubation time exceeded 1 h, all the tubes turned red, indicating a positive response, therefore the incubation time should not exceed 30 min. Fourteen different isolates were found to be PUFA producers, either producing EPA or DHA or both. Eleven of these were isolated from the Mid-Atlantic Ridge sample while the rest were isolated from the Red Sea. Mediterranean Sea isolates showed no sign of the ability to produce PUFAs. This inability to produce PUFAs could be due to the high level of pollution compared to the Red Sea. The United Nations Environment Programme has estimated that 650,000,000 t of sewage are disposed in the Mediterranean sea yearly [29], while the Red Sea is treated by the Egyptian government as an environmentally protected area for tourism. In a comparative study, [29] reported that the concentration of iron in the Mediterranean Sea was double that in the Red Sea. In our preliminary results (data not shown), iron showed a significant negative impact on the ability of the isolates under investigation to produce PUFAs. The other possibility for the absence of PUFA producers in the Mediterranean Sea may be that the salinity of the sea is much lower compared to the Red Sea, as the ability to produce PUFAs is reported to be a response to the exposure to environmental stress. The Red Sea salinity is 4 % higher than the average level of salinity in seas and oceans. Also the Red Sea proved to be populated with a high number of extremophiles due to the high environmental stress [30]. The analysis of the PB screening experiment showed that four variables have a significant effect on all the EPA responses namely glycerol, whey, NaCl and Na2HPO4. The tested sugars, including maltose, lactose and mannitol, showed either no significant effect or a statistically significant negative effect on the ability to produce EPA compared to the other tested factors. Sodium chloride was found to have a statistically significant influence on the EPA production by isolate 66 with the minimum concentration of NaCl (10 g/l) resulting in higher production. Low concentration of sodium chloride in the production medium minimises the potential corrosion issues in the large-scale production. In addition, the negative effect of the high concentration of sodium chloride was previously reported on the DHA producer Crythecodinium cohnii ATCC 30556, with a significant decrease in the amount of DHA produced when doubling the concentration of sodium chloride from 15 to 30 g/l in the cultivation media [31].

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Na2HPO4 showed a statistically significant positive effect on the ability of isolate 66 to produce EPA which could be due to the effect of phosphate. This is in line with the observations of [9] where increasing the amount of KH2PO4, as a source of inorganic phosphate in the culture media of Thraustochytrium sp., from 0.1 to 0.2 g/l led to a 40 % increase in the DHA yield. A potential economically viable medium for EPA production was investigated in a separate CCD experiment. Glycerol and whey were considered as components of this potential production medium, as they are by-products produced during other industrial processes. A production medium consisting of such cheap components, which would otherwise have to be disposed of, offers a number of advantages from the point of view of economically viable manufacture of valuable products, such as PUFAs. The main by-product of the biodiesel industry is glycerol, which usually requires costly purification steps. Glycerol has been used as a carbon source for a range of microbial fermentation processes to produce different products including lipids [32, 33]. The use of glycerol as a potential medium component for PUFA production was previously reported by [34]. With EPA present in the bacterial cells in the form of phospholipids, glycerol may be significant due to the fact that it is the main backbone of phosphatidic acid which is the key precursor for the biosynthesis of phospholipid headgroup structures [35]. Whey is considered as the main by-product of the dairy industry and is produced in massive quantities [36]. Recently, cheese whey was successfully used to support the growth and γ-linolenic acid (GLA) production by Mortierella isabellina achieving 301 mg/l [37].

Conclusion The abundance of PUFA producers in the cold environment reinforces the argument that they are required to sustain the fluidity of the plasma membrane under such conditions. The Mediterranean Sea and the Red Sea were screened for PUFA producers for the first time and the obtained results indicated that Red Sea could be a potential source of microbial PUFAs, although more work is required. The colourimetric method for screening PUFAs producers was found to have a high falsepositive result but zero negative false result which may lead to decreasing the effort and time required to screen a large number of isolates. By applying Plackett–Burman and response surface methodology, the amount of EPA yield increased from 5.44 mg/g, 7.5 mg/l representing 3.5 % of the total fatty acids to 20 mg/g, 45 mg/l representing 11 % of the total fatty acids. The ability to produce EPA as a sole PUFA which could be advantageous from the downstream processing prospective in addition to the high productivity on cheap industrial by-products make the

Screening of Marine Bacterial Producers of Polyunsaturated Acids

isolate 66 a potential high producer in the industrial scale making the microbial production of EPA more feasible. The isolate under investigation can be considered a high EPA producer producing even higher levels than a genetically engineered cyanobacterium, Synechococcus sp. which is able to produce 7.6 mg/l [38].

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16.

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