Response surface methodology for optimising the culture conditions for eicosapentaenoic acid production by marine bacteria

Polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA), are increasingly attracting scientific attention owing to their significant health-promoting role in the human body. However, the human body lacks the ability to produce them in vivo. The limitations associated with the current sources of ω-3 fatty acids from animal and plant sources have led to increased interest in microbial production. Bacterial isolate 717 was identified as a potential high EPA producer. As an important step in the process development of the microbial PUFA production, the culture conditions at the bioreactor scale were optimised for the isolate 717 using a response surface methodology exploring the significant effect of temperature, pH and dissolved oxygen and the interaction between them on the EPA production. This optimisation strategy led to a significant increase in the amount of EPA produced by the isolate under investigation, where the amount of EPA increased from 9 mg/g biomass (33 mg/l representing 7.6 % of the total fatty acids) to 45 mg/g (350 mg/l representing 25 % of the total fatty acids). To avoid additional costs associated with extreme cooling at large scale, a temperature shock experiment was carried out reducing the overall cooling time from the whole cultivation process to 4 h only prior to harvest. The ability of the organism to produce EPA under the complete absence of oxygen was tested revealing that oxygen is not critically required for the biosynthesis of EPA but the production improved in the presence of oxygen. The stability of the produced oil and the complete absence of heavy metals in the bacterial biomass are considered as an additional benefit of bacterial EPA compared to other sources of PUFA. To our knowledge this is the first report of a bacterial isolate producing EPA with such high yields making the large-scale manufacture much more economically viable.     

Screening of Marine Bacterial Producers of Polyunsaturated Fatty Acids and Optimisation of Production

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 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.     

Candida zeylanoides as a new yeast model for lipid metabolism studies: effect of nitrogen sources on fatty acid accumulation

Lipid homeostasis is well-known in oleaginous yeasts, but there are few non-oleaginous yeast models apart from Saccharomyces cerevisiae. We are proposing the non-oleaginous yeast Candida zeylanoides QU 33 as model. The aim of this study was to investigate the influence of the carbon/nitrogen ratio and the type of nitrogen source upon oil accumulation by this yeast grown on shake flask cultures. The maximum biomass was obtained in yeast extract (2.39?±?0.19 g/l), followed by peptone (2.24?±?0.05 g/l), while the highest content of microbial oil (0.35?±?0.01 g/l) and the maximum lipid yield (15.63 %) were achieved with peptone. Oleic acid was the predominant cellular fatty acid in all culture media (>32.23 %), followed by linoleic (>15.79 %) and palmitic acids (>13.47 %). The highest lipid yield using glucose and peptone was obtained at the C/N ratio of 200:1.     

Optimization of the culture condition for an antitumor bacterium Serratia proteamacula 657 and identification of the active compounds

Exploration of novel active anti-tumor compounds from marine microbes for pharmaceutical applications has been a continuously hot spot in natural product research. Bacterial growth and metabolites may greatly vary under different culture conditions. In this study, the effects of different culture conditions and medium components on the growth and bioactive metabolites of Serratia proteamacula 657, an anti-tumor bacterium found in our previous study, were investigated. The results showed that lower temperature, weak acidic condition and solid fermentation favored the bacterial growth and the production of active compounds. Four components in the culture medium, NaCl, peptone, yeast extract and MgSO₄, were found important to the bacterial growth and active compounds production in medium optimization. Under the optimized condition of solid state fermentation at pH 6.0–7.0, 23–25 °C, with the MgSO₄-free medium containing 10.0 g/L peptone, 1.0 g/L yeast extract and 19.45 g/L NaCl, the antitumor activity of S. proteamacula 657 and the yield of crude extracts increased about 15 times and 6 times than the sample obtained in the original liquid fermentation, respectively. The active components in the metabolites of S. proteamacula 657 were identified as a homolog of prodigiosin, a red bacterial pigment, based on the analysis of the NMR and GC–MS. The bacterium S. proteamacula 657, which is adapted to lower temperature, produced prodigiosin-like pigments with highly antitumor activity, suggesting the bacterium is a potential new source for prodigiosin production.     

Process development of eicosapentaenoic acid production

Eicosapentaenoic acid (EPA), a well-known member of omega-3 fatty acids, is considered to have a significant health promoting role in the human body. It is an essential fatty acid as the human body lacks the ability to produce it in vivo and must be supplemented through diet. Microbial EPA represents a potential commercial source. GC/MS analyses confirmed that bacterial isolate 717, similar to Shewanella pacifica on the basis of 16S rRNA sequencing, is a potential high EPA producer. Two types of bioreactors, a Stirred Tank Reactor (STR) and an Oscillatory Baffled Reactor (OBR), were investigated in order to choose the optimum system for EPA production. The EPA production media was optimised through the selection of media components in a Plackett–Burman (PB) design of experiment followed by a Central Composite Design (CCD) to optimise the concentration of medium components identified as significant in the Plackett–Burman experiment. The growth conditions for the bioreactor, using artificial sea water (ASW) medium, were optimised by applying Response Surface Methodology (RSM). This optimisation strategy resulted in an increase in EPA from 33 mg/l (10 mg/g biomass), representing 8% of the total fatty acids at shake flask level, to 350 mg/l (46 mg/g biomass) representing 25% of the total fatty acids at bioreactor level. During this study the main effects and the interactions between the bioreactor growth conditions were revealed and a polynomial model of EPA production was generated. Chemostat experiments were performed to test the effect of growth rate and temperature on EPA production.     

二十碳五烯酸的菌种选育及发酵条件

利用含亚麻子油的斜面培养基连续传代和逐渐降低培养温度,诱导筛选的方法,从大量丝状真菌中选育到一株产二十碳五烯酸（all－cis－5、8、11、14、17－eicosopnthenoicacid）较高的被孢霉菌（Motierellasp．）SM481。研究得到最适培养基及最适培养条件。在最适培养及产二十碳五烯酸条件下,细胞干重和二十碳五烯酸产量分别为28．8g／L和0.127g／L。     

Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis

An isolate from kimchi, identified as Lactobacillus brevis, accumulated γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in the culture medium. Optimal culture conditions for growth of L. brevis and production of GABA were 6 % (w/v) l-glutamic acid, 4 % (w/v) maltose, 2 % (w/v) yeast extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl₂, 2 g Tween 80/l, and 0.02 mM pyridoxal 5′-phosphate at initial pH 5.25 and 37 °C. GABA reached 44.4 g/l after 72 h cultivation with a conversion rate 99.7 %, based on the amount (6 %) of l-glutamic acid added. GABA was purified using ion exchange column chromatography with 70 % recovery and 97 % purity.     

Heavy oils,principally long-chain n-alkanes secreted by Aureobasidium pullulans var. melanogenum strain P5 isolated from mangrove system

In this study, the yeast strain P5 isolated from a mangrove system was identified to be a strain of Aureobasidium pullulans var. melanogenum and was found to be able to secrete a large amount of heavy oil into medium. After optimization of the medium for heavy oil production and cell growth by the yeast strain P5, it was found that 120.0 g/l of glucose and 0.1 % corn steep liquor were the most suitable for heavy oil production. During 10-l fermentation, the yeast strain P5 produced 32.5 g/l of heavy oil and cell mass was 23.0 g/l within 168 h. The secreted heavy oils contained 66.15 % of the long-chain n-alkanes and 26.4 % of the fatty acids, whereas the compositions of the fatty acids in the yeast cells were only C_16:0 (21.2 %), C_16:1(2.8 %), C_18:0 (2.9 %), C_18:1 (39.8 %), and C_18:2 (33.3 %). We think that the secreted heavy oils may be used as a new source of petroleum in marine environments. This is the first report of yeast cells which can secrete the long-chain n-alkanes.     

Eicosapentaenoic and docosahexaenoic acids production by and okara-utilizing potential of thraustochytrids

Nine thraustochytrid strains isolated from subtropical mangroves were screened for their eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) production potential in a glucose yeast extract medium. Their ability to utilize okara (soymilk residue) for growth and EPA and DHA production was also evaluated. EPA yield was low in most strains, while DHA level was high on glucose yeast extract medium, producing 28.1–41.1% of total fatty acids, for all strains, with the exception of Ulkenia sp. KF13. The DHA yield of Schizochytrium mangrovei strains ranged from 747.7 to 2778.9 mg/l after 52 h of fermentation at 25°C. All strains utilized okara as a substrate for growth, but DHA yield was lower when compared with fermentation in a glucose yeast extract medium. Journal of Industrial Microbiology & Biotechnology (2001) 27, 199–202. Received 11 December 2000/ Accepted in revised form 29 June 2001     

Augmented cellulase production by Bacillus subtilis strain MU S1 using different statistical experimental designs

The production of cellulase by Bacillus subtilis MU S1, a strain isolated from Eravikulam National Park, was optimized using one-factor-at-a-time (OFAT) and statistical methods. Physical parameters like incubation temperature and agitation speed were optimized using OFAT and found to be 40?°C and 150?rpm, respectively, whereas, medium was optimized by statistical tools. Plackett-Burman design (PBD) was employed to screen the significant variables that highly influence cellulase production. The design showed carboxymethyl cellulose (CMC), yeast extract, NaCl, pH, MgSO₄ and NaNO₃ as the most significant components that affect cellulase production. Among these CMC, yeast extract, NaCl and pH showed positive effect whereas MgSO₄ and NaNO₃ were found to be significant at their lower levels. The optimum levels of the components that positively affect enzyme production were determined using response surface methodology (RSM) based on central composite design (CCD). Three factors namely CMC, yeast extract and NaCl were studied at five levels whilst pH of the medium was kept constant at 7. The optimal levels of the components were CMC (13.46?g/l), yeast extract (8.38?g/l) and NaCl (6.31?g/l) at pH 7. The maximum cellulase activity in optimized medium was 566.66?U/ml which was close to the predicted activity of 541.05?U/ml. Optimization of physical parameters and medium components showed an overall 3.2-fold increase in activity compared to unoptimized condition (179.06?U/ml).     

Production of lactic acid and fungal biomass by Rhizopus fungi from food processing waste streams

This study proposed a novel waste utilization bioprocess for production of lactic acid and fungal biomass from waste streams by fungal species of Rhizopus arrhizus 36017 and R. oryzae 2062. The lactic acid and fungal biomass were produced in a single-stage simultaneous saccharification and fermentation process using potato, corn, wheat and pineapple waste streams as production media. R. arrhizus 36017 gave a high lactic acid yield up to 0.94–0.97 g/g of starch or sugars associated with 4–5 g/l of fungal biomass produced, while 17–19 g/l fungal biomass with a lactic acid yield of 0.65–0.76 g/g was produced by the R. oryzae 2062 in 36–48 h fermentation. Supplementation of 2 g/l of ammonium sulfate, yeast extract and peptone stimulated an increase in 8–15% lactic acid yield and 10–20% fungal biomass.

     

Fatty acid contents and profiles of 16 macroalgae collected from the Irish Coast at two seasons

Due to the established health benefits of omega-3 long-chain polyunsaturated fatty acids (LC-PUFA), there is a globally increasing demand for alternative natural resources with appropriate fatty acid profiles. To assess the suitability of macroalgae as a source, 16 species (nine Phaeophyceae, five Rhodophyta and two Chlorophyta) were collected at two seasons (June and November) from the Irish west Coast, and total fatty acid contents and specific profiles were determined. Total fatty acid contents, expressed per percentage of dry weight, ranged from 6.4 %?±?0.3 (Pelvetia canaliculata, Phaeophyceae) to 0.8 %?±?0.2 (Porphyra dioica, Rhodophyta). Most common fatty acids were palmitic (16:0), oleic (OLE, 18:1 n-9), α-linolenic (ALA, 18:3 n-3), arachidonic (ARA, 20:4 n-6) and eicosapentaenoic (EPA, 20:5 n-3) acids. Fatty acid profiles were highly variable between and within algal groups; red and brown seaweeds were generally richer in LC-PUFA (e.g. 20:4 n-6 and 20:5 n-3), while high levels of saturated fatty acids such as palmitic acid (16:0) were observed in green species. Most omega-3 PUFA-rich species investigated had a omega-6/omega-3 fatty acid ratio close to 1, which is favourable for human health. The two seasonal sampling times revealed significant differences in total fatty acid and 20:5 n-3 (EPA) contents, with changes depending on species, thus implying varying suitability as potential target species for EPA production. At both times of the year, Palmaria palmata was identified as most promising species as a source of 20:5 n-3 (EPA) amongst all species investigated, with levels ranging from 0.44 to 0.58 % of dry weight in June and November, respectively.     

Statistical optimization of lovastatin production by Omphalotus olearius (DC.) singer in submerged fermentation

In this study, culture conditions were optimized to improve lovastatin production by Omphalotus olearius, isolate OBCC 2002, using statistical experimental designs. The Plackett–Burman design was used to select important variables affecting lovastatin production. Accordingly, glucose, peptone, and agitation speed were determined as the variables that have influence on lovastatin production. In a further experiment, these variables were optimized with a Box–Behnken design and applied in a submerged process; this resulted in 12.51 mg/L lovastatin production on a medium containing glucose (10 g/L), peptone (5 g/L), thiamine (1 mg/L), and NaCl (0.4 g/L) under static conditions. This level of lovastatin production is eight times higher than that produced under unoptimized media and growth conditions by Omphalotus olearius. To the best of our knowledge, this is the first attempt to optimize submerged fermentation process for lovastatin production by Omphalotus olearius.     

Phosphate limitation promotes unsaturated fatty acids and arachidonic acid biosynthesis by microalgae <Emphasis Type="Italic">Porphyridium purpureum</Emphasis>

Polyunsaturated fatty acids (PUFAs) are highly appreciated on their nutritive value for human health and aquaculture. P. purpureum, one of the red microalgae acknowledged as a promising accumulator of ARA, was chosen as the target algae in the present research. Effects of sodium bicarbonate (0.04–1.2 g/L), temperature (25, 30 and 33 °C) and phosphate (0.00–0.14 g/L) on biomass yield, total fatty acids (TFA) and arachidonic acid (ARA) accumulation were investigated systemically. NaHCO₃ dose of 0.8 g/L and moderate temperature of 30 °C were preferred. In addition, TFA and ARA production were significantly enhanced by an appropriate concentration of phosphate, and the highest TFA yield of 666.38 mg/L and ARA yield of 159.74 mg/L were obtained at a phosphate concentration of 0.035 g/L. Interestingly, with phosphate concentration continuing to fall, UFA/TFA and ARA/EPA ratios were increased accordingly, suggesting that phosphate limitation promoted unsaturated fatty acids and arachidonic acid biosynthesis. Low concentration of phosphate may be favored to increase the enzymatic activities of ?6-desaturase, which played a key role in catalyzing the conversion of C16:0 to C18:2, and thus the selectivity of UFA increased. Meanwhile, the increase of ARA selectivity could be attributed to ω6 pathway promotion and ?17-desaturase activity inhibition with phosphate limitation. Phosphate limitation strategy enhanced unsaturated fatty acids and ARA biosynthesis in P. purpureum, and can be applied in commercial scale manufacturing and commercialization of ARA.     

Extractability of polyhydroxyalkanoate synthesized by <Emphasis Type="Italic">Bacillus flexus</Emphasis> cultivated in organic and inorganic nutrient media

Bacillus flexus was isolated from local soil sample and identified by molecular methods. In inorganic nutrient medium (IM) containing sucrose as carbon source, yield of biomass and polyhydroxyalkanoate (PHA) were 2 g/l and 1 g/l (50% of biomass), respectively. Substitution of inorganic nitrogen by peptone, yeast extract or beef extract resulted in biomass yields of 4.1, 3.9 and 1.6 g/l, respectively. Corresponding yields of PHA in biomass was 30%, 40% and 44%. Cells subjected to change in nutrient condition from organic to inorganic, lacked diaminopimelic acid in the cell wall and the concentration of amino acids also decreased. Under these conditions the extractability of the polymer from the cells by hot chloroform or mild alkali hydrolysis was 86–100% compared to those grown in yeast extract or peptone (32–56%). The results demonstrated that growth, PHA production and the composition of cell wall of B. flexus are influenced by the organic or inorganic nutrients present in the growth medium. Cells grown in inorganic medium lysed easily and this can be further exploited for easier recovery of the intracellular PHA.     

Ethanol production from acid- and alkali-pretreated corncob by endoglucanase and β-glucosidase co-expressing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> subject to the expression of heterologous genes and nutrition added

Low-cost technologies to overcome the recalcitrance of cellulose are the key to widespread utilization of lignocellulosic biomass for ethanol production. Efficient enzymatic hydrolysis of cellulose requires the synergism of various cellulases, and the ratios of each cellulase are required to be regulated to achieve the maximum hydrolysis. On the other hand, engineering of cellulolytic Saccharomyces cerevisiae strains is a promising strategy for lignocellulosic ethanol production. The expression of cellulase-encoding genes in yeast would affect the synergism of cellulases and thus the fermentation ability of strains with exogenous enzyme addition. However, such researches are rarely reported. In this study, ten endoglucanase and β-glucosidase co-expressing S. cerevisiae strains were constructed and evaluated by enzyme assay and fermentation performance measurement. The results showed that: (1) maximum ethanol titers of recombinant strains exhibited high variability in YPSC medium (20 g/l peptone, 10 g/l yeast extract, 100 g/l acid- and alkali-pretreated corncob) within 10 days. However, they had relatively little difference in USC medium (100 g/l acid- and alkali-pretreated corncob, 0.33 g/l urea, pH 5.0). (2) Strains 17# and 19#, with ratio (CMCase to β-glucosidase) of 7.04 ± 0.61 and 7.40 ± 0.71 respectively, had the highest fermentation performance in YPSC. However, strains 11# and 3# with the highest titers in USC medium had a higher ratio of CMCase to β-glucosidase, and CMCase activities. These results indicated that nutrition, enzyme activities and the ratio of heterologous enzymes had notable influence on the fermentation ability of cellulase-expressing yeast.     

Enhanced production of amidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> MTCC 1526 by medium optimisation using a statistical experimental design

In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R. erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket–Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g dry cells, respectively. Thus, use of RSM increased production of amidase from R. erythropolis MTCC 1526 by 6.88-fold.     

Joint effect of nitrogen sources and B vitamin supplementation of date juice on lactic acid production by Lactobacillus casei subsp. rhamnosus

The use of date juice as a substrate for lactic acid production was investigated. Various nitrogen sources were compared with yeast extract for efficient lactic acid production by Lactobacillus casei subsp. rhamnosus. Among different nitrogen sources added to date juice (yeast extract, ammonium sulfate, tryptic soy, urea, peptone, and casein hydrolysate), yeast extract was the most efficient. The effect of yeast extract could have been due to its B vitamin content. The addition of five B vitamins at less than 25 mg/l to date juice with any nitrogen source enhanced lactic acid production to some extent, except for date juice with yeast extract or urea or peptone. The most significant increase was obtained with ammonium sulfate. Half of the yeast extract content (10 g/l) in a supplemented date juice could be replaced by a mixture of B vitamins at less than 25 mg/l, and ammonium sulfate at 2.6 g/l with no significant decrease in lactic acid production.     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Efficient preparation of pseudoalteromone A from marine Pseudoalteromonas rubra QD1-2 by combination of response surface methodology and high-speed counter-current chromatography: a comparison with high-performance liquid chromatography

Pseudoalteromone A (PA) is a cytotoxic and anti-inflammatory ubiquinone discovered recently from a marine bacterium Pseudoalteromonas sp. CGH2XX. In order to meet its sample supply for further in vivo pharmacological investigation, an efficient method was developed for the preparation of PA by combination of response surface methodology (RSM) and high-speed counter-current chromatography (HSCCC) from marine bacterium P. rubra QD1-2. First, optimization of culture conditions was studied by the RSM to enhance PA production. The results indicated that the optimal cultivation condition was peptone (2.21 g/l), yeast extract (3.125 g/l), glucose (0.125 g/l), KBr (0.02 g/l), inoculum size (6.5 %), medium volume (595 ml), initial pH value (7.0), temperature (28 °C). Under the optimized fermentation condition, PA production was 1.04 mg/l with 14.8-fold increase comparing to 0.07 mg/l under original standard fermentation condition. The PA production was further investigated using a 14-l jar fermenter. Compared to the flask culture, P. rubra QD1-2 offered 45 % increase of PA production at 1.51 mg/l. Then, a rapid and efficient method for the separation and purification of PA from crude culture extract was developed using HSCCC. The two-phase solvent system used for HSCCC separation was composed of n-hexane–ethyl acetate–methanol–water (5:5:9:5, v/v/v/v). The isolation was accomplished within 100 min, and the purity of PA was over 95 %. The recovery of the process was 93 %.