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Optimization of medium for ascochlorin production by the leafhopper pathogenic fungus Microcera sp. BCC 17074.

Ascochlorin is an isoprenoid antibiotic structurally related to ubiquinol (Figure 1). It was first isolated from Ascochyta viciae LIBERT by Tamura et al. (1) and Sasaki et al. (2) and was described as antiviral agent. Ascochlorin has also been isolated from several other genera such as Nectria (3), Acremonium (4), Verticillium (5), Cylindrocarpon (6) and Microcera (7), among others. Ascochlorin is an unusual cytochome [bc.sub.1] inhibitor that acts at both of the active sites of the enzymes [Q.sub.o] and [Q.sub.i]. It is a specific inhibitor that acts on mitochondria (8).

Ascochlorin has also shown antiviral and antitumor activities (1, 9). Interestingly, significant effects on in vivo breast cancer propagation could be shown using this compound (10). Effects on hypertension were also demonstrated (11).

Our collaborative research group at the National Center for Genetic Engineering and Biotechnology (Thailand) recently reported the isolation of ascochlorin and related compounds from cultures of the leafhopper pathogenic fungus Microcera sp. BCC 17074 (7). With this strain, ascochlorin was produced as a major secondary metabolite with a low amount of other compounds. The yield of ascochlorin using BCC 17074, based on the volume of the fermentation medium, was a relatively efficient level when compared with the data from experimental procedures in other ascochlorin isolation papers, in which a yield of 37 mg was obtained from Verticillium hemipterigenum BCC 2370 (5). The goal of this project was to increase the productivity of ascochlorin using our strain and to confirm the results in the bioreactor to demonstrate the applicability of the procedure for production. A full factorial design was used to determine the optimal carbon and nitrogen sources, which allows for a clear selection of the best carbon and nitrogen sources. Following this selection, a fractional factorial design at 2 levels (Plackett-Burman design) was used for the influential quantitative factors, and biomass and ascochlorin production were taken into consideration as secondary responses in order to evaluate the relationship between growth and production. The last step of optimization was performed using a central composite design to obtain the optimal range of the most influential factors, and then the optimal conditions were confirmed in a 5-L bioreactor.

EXPERIMENTAL

Materials and Methods Fungal strain

The fungus used in this study, Microcera sp., is preserved at the BIOTEC Culture Collection (BCC) as BCC 17074.

Inoculum

The fungus was initially grown on potato dextrose agar (PDA) at 25[degrees]C for 3-5 days. An agar block of 1 [cm.sup.3] containing Microcera sp. BCC 17074 was cut into small pieces, transferred to a 250 ml Erlenmeyer flask containing 50 ml of potato dextrose broth (Becton, Dickinson and company, MD, USA) and incubated at 25[degrees]C for 3-5 days on a rotary shaker at a shaking speed of 200 rpm (New Brunswick, NJ, USA). This primary culture was transferred to a 1 L Erlenmeyer flask containing 250 ml of PDB, incubated at 25[degrees]C for 3-5 days on a rotary shaker at a shaking speed of 200 rpm and was used as the inoculum in all experiments. Experimental design

A general factorial design was used to determine the optimal carbon and nitrogen sources. The optimization was performed in 250 mL Erlenmeyer flask containing 50 mL of medium. Approximately 20 g/L of 6 different carbon sources (maltose, trehalose, glucose, fructose, galactose and sorbitol) and 4 g/L of 5 nitrogen sources (ammonium chloride, malt extract (Becton, Dickinson and company, MD, USA), yeast extract (Becton, Dickinson and company, MD, U SA), meat extract (Merck KGaA, Damstadt, Germany) and tryptone (Becton, Dickinson and company, MD, USA)) were used. When the optimal carbon and nitrogen sources were obtained, a two-level fractional factorial design of [2.sup.n-1] (Plackett-Burman design) was applied to 9 selected factors influencing ascochlorin production: fructose (2060 g/L), yeast extract (2-6 g/L), ammonium chloride (0-4 g/L), NaCl (0-2 g/L), Ca[Cl.sub.2][double dagger]2[H.sub.2]O (0-0.2 g/L), K[H.sub.2]P[O.sub.4](0-2 g/L), [K.sub.2]HP[O.sub.4](0-2 g/L), KCl (0-2 g/L) and trace elements (0-2 mL/L). Experiments were conducted in duplicate and with 3 center points using a fold-over augment (27 runs and 3 center points). When the factors from the Plackett-Burman design were selected, a response surface of the central composite design was conducted with 5 selected factors influencing ascochlorin production: fructose (20-60 g/L), yeast extract (2-6 g/L), ammonium chloride (2-4 g/L), NaCl (2-4 g/L) and K[H.sub.2]P[O.sub.4] (2-4 g/L), with a practical alpha factor (1.49535) and a small size of central composite design, were used. A quadratic model was obtained allowing the determination of the levels of the 5 selected nutritional factors. All results were analyzed with Design Expert software (Version 7.0.b1.1, Stat-Ease Inc., Minneapolis, USA). Fermentation condition

For scale-up studies, the fungus fermentation was conducted in a 5-L bioreactor (B.E. Marubishi, Pathum Thani, Thailand). The fermenter equipped with 6-blade turbine, baffled pyrex jacket, and round shape sparger. The medium used a working volume of 4 L containing 40 g/L fructose, 4 g/L yeast extract, 3 g/L ammonium chloride, 3 g/L NaCl and 4 g/L K[H.sub.2]P[O.sub.4]. The cultivation was performed at 25[degrees]C, an agitation speed of 300 rpm, an aeration rate of 1 vvm and a pH that was not controlled.

Biomass determination

Biomass content was determined by harvesting 1.6 ml of sample, which was centrifuged at 12000 rpm for 2 min. The filter cakes were washed with distilled water and dried at 105-110[degrees]C for 2448 h until a stable weight was achieved. The culture filtrate was then subjected to metabolite and sugar analyses.

Ascochlorin extraction and quantification

The culture broth was harvested and filtered using Whatman No 1 filter paper. The filter cake was immersed in 40 ml of methanol for 24 h. The extracted supernatant was then filtered through Whatman No 1 filter paper. The filtrate was poured into a separation funnel, an equal volume of hexane was added and the layers were separated. The bottom (methanol) layer was evaporated, and the residue was diluted with distilled water (25 mL) and extracted with ethyl acetate (3 x 25 mL). The combined organic layer was concentrated under reduced pressure to obtain the crude mycelial extract, which was subjected to HPLC analysis for quantification of ascochlorin. The extract was dissolved in 1 ml of methanol, and a 20 mL portion was injected. Ascochlorin was detected using HPLC employing a reverse phase NovaPak C18 column, 3.9x150 mm with a 5 mm particle size (Waters, MA, USA) and acetonitrile:methanol,:0.05% TFA in water (Composition 25:25:50) as mobile phases at a flow rate of 0.45 mL/min, monitored spectrophotometrically at 220 nm (Waters 996 Photodiode Array Detector). Standard ascochlorin was isolated and purified from the same fungal strain (7). The concentration of ascochlorin was determined from a standard curve (0 to 1000 mg/L).

The retention time of ascochlorin was at 6.3-6.4 min.

Sugar determination

The medium was centrifuged at 12,000 rpm for 2 min, and the supernatant was filtered through a 0.22 [micro]m MCM syringe filter. The filtrate was subjected to HPLC analysis using an Aminex Resin-Based column with 5 mM sulfuric acid as a mobile phase at a flow rate of 0.6 ml x [min.sup.-1]. Sugars were detected refractometrically with a Waters 2414 Refractive Index Detector. A standard curve for sugar determination was prepared by using a standard sugar solution in the concentration range of 1.25-20.0 g/L.

RESULTS

The productions of biomass and ascochlorin by Microcera sp. BCC 17074 were evaluated on different carbon and nitrogen sources using a general factorial design: 6 carbon sources and 5 nitrogen sources were used. The model of ascochlorin production on different carbon and nitrogen sources are shown in equations 1 and 2, respectively. Fructose and yeast extract had the highest positive effects on ascochlorin production, respectively.

Ascochlorin (mg/L)=5.09-3.08A+1.81B+2.57C 1.16D-0.16E-3.05F-3.27G-3.29H+5.00I+3.88J-2.51K ... (1)

and

biomass (g/L) =+5.89-0.51A+0.92B+0.73C-0.33D0.01E-1.61F-4.68G-3.32H+4.14I+3.64J+0.22K ...(2)

where A-maltose, B-trehalose, Cfructose, D-galactose, E-glucose, F-sorbitol, G ammonium chloride, H-malt extract, I-yeast extract, J-meat extract and K-tryptone.

The highest biomass production of 12.6 [+ or -] 2.40 g/L was obtained on a fructose/meat extract, while the highest ascochlorin production of 20.19 [+ or -] 0.01 mg/L was obtained on a fructose/ yeast extract (Table 1). The highest ascochlorin yield of 2.08 [+ or -] 0.01 mg/g DW was obtained on trehalose and ammonium chloride. The analysis of variance of the general factorial design for biomass and ascochlorin production by Microcera sp. BCC 17074 is shown in Tables 2 and 3. After this selection, biomass and ascochlorin production were then subjected to a Plackett-Burman design using 9 factors. The influence of two levels of fructose and yeast extract along with chloridecontaining compounds (N[H.sub.4]Cl, Ca[Cl.sub.2] x 2[H.sub.2]O, NaCl and KCl) was evaluated on ascochlorin production. The highest biomass production of 23.40 [+ or -] 1.84 g/L was obtained on 60 g/L fructose, 6 g/L yeast extract, 2 g/L NaCl and 2 g/L [K.sub.2]HP[O.sub.4], but there was no production of ascochlorin. In contrast, the highest ascochlorin production of 24.60 [+ or -] 0.76 mg/L was obtained on 60 g/L fructose, 6 g/L yeast extract, 2 g/L NaCl, 0.2 g/L Ca[Cl.sub.2] x 2[H.sub.2]O and 2 g/L K[H.sub.2]P[O.sub.4], in which 15.75 [+ or -] 0.21 g/L of biomass was obtained. The highest ascochlorin yield of 1.86 [+ or -] 0.22 mg/g DW was obtained on 60 g/L fructose, 6 g/L yeast extract, 4 g/L NaCl, 2 g/L [K.sub.2]HP[O.sub.4] and 2 mL/L of trace elements solution (Table 4). The models of ascochlorin and biomass production are shown in equation 3 and 4, in which fructose, yeast extract, ammonium chloride, NaCl and K[H.sub.2]P[O.sub.4] were the most positive influential factors on the production of ascochlorin. The standard deviation of 4.60 and [R.sup.2] of 0.90 were obtained for ascochlorin production, while curvature and the lack of fit were not significant (Table 5 and 6).

Ascochlorin (mg/L) =6.15+2.81A+0.67B+ 2.35C+0.72D-0.043E+1.02F-0.39G-0.64H+0.076J ...(3)

and

biomass (g/L) = 10.66+1.79A+2.44B+1.00C+0.91D0.65E-0.11F0.21G-0.45H-0.91J ... (4)

where A-fructose, B-yeast extract, Cammonium chloride, D-NaCl, E-Ca[Cl.sub.2].2[H.sub.2]O, FK[H.sub.2]P[O.sub.4], G-[K.sub.2]HP[O.sub.4], H-KCl and J-trace elements.

The optimization was carried out using a central composite design with the 4 influential factors, and the addition of N[H.sub.4]Cl. N[H.sub.4]Cl was chosen to lower the cost of yeast extract and also because the chloride ion appeared to affect ascochlorin production (Figure 2). The highest biomass production of 19.90 [+ or -] 0.78 g/L was obtained on 60 g/L fructose, 6 g/L yeast extract, 4 g/L N[H.sub.4]Cl, 2 g/L NaCl and 2 g/L K[H.sub.2]P[O.sub.4], whereas the highest ascochlorin production and yield of 41.12 [+ or -] 0.62 mg/ L and 2.19 [+ or -] 0.03 g/g DW, respectively, were obtained on 40 g/L fructose, 4 g/L yeast extract, 3 g/L N[H.sub.4]Cl, 3 g/L NaCl and 4 g/L K[H.sub.2]P[O.sub.4] (Table 7). The models of ascochlorin and biomass production obtained from the central composite design are shown in equation 5 and 6, with sodium chloride and yeast extract having the highest positive effects on the production of ascochlorin and biomass, respectively. The interaction between yeast extract/ammonium chloride and fructose/ yeast extract had the highest positive effects on ascochlorin and biomass production, respectively. The standard deviation of 6.94 and an R2 of 0.95 were obtained for ascochlorin production, and the lack of fit was not significant (Table 8 and 9).

Ascochlorin (mg/L) =18.48+0.018A+2.92B +0.94C+8.29D+3.99E+11.62AB + 10.16AC-0.58AD+3.67AF+4.70BC-4.59BD0.10BF-4.18CD0.30CF-8.66DF-8.97A2-4.60B21 68C2-3 09D2+6.72F2 ... (5) and biomass (g/L) = 13.44+0.47A+2.78B 0.17C+0.32D+1.47F+2.44AB+2.15AC +2.22AD+0.72AF-0.87BC-L03BD-2.59BF-0.90CD2.26CF-1.62DF-4.24 A2 -0.64B2+0.75C2+ 1.63D2+1.58F2 ... (6) where A-fructose, B-yeast extract, C-ammonium chloride, D-NaCl and F-K[H.sub.2]P[O.sub.4].

The model was then confirmed in a 5-L bioreactor with the selected optimized medium obtained from the central composite design; the highest biomass production of 19.6 [+ or -] 0.99 g/L was obtained at 193 h, and the highest ascochlorin production of 68.35 [+ or -] 1.17 mg/L was achieved at 144 h (Figure 3).

DISCUSSION

Microcera sp. BCC 17074 produced ascochlorin as a major compound unlike Verticillium hemipterigenum BCC 2370, which produced a lower level of ascochlorin together with other ascochlorin derivatives (5, 7). Chloride ions directly affected the production of ascochlorin produced by Microcera sp. BCC 17074, as the ascochlorin structure contains chloride on the benzene ring (1,7). Because ascochlorin is a chlorine atomcontaining molecule, it was suspected that the addition of a chloride ion source to the medium would enhance the production of this compound by Microcera sp. BCC 17074. The present results were consistent with this hypothesis. Thus, increased the levels of N[H.sub.4]Cl and NaCl concentrations resulted in the higher production of ascochlorin, and the results showed that the enriched chloride-containing compounds in the medium enhanced the ascochlorin production. The production of ascochlorin by Microcera sp. BCC 17074 in a 5-L bioreactor (68.35 [+ or -] 1.17 mg/L) was higher compared with the screening medium (20.19 [+ or -] 0.01 mg/L) and was 1.9 times higher than that from Verticillium hemipterigenum BCC 2370 (37 mg/L) (5). Similar findings were reported on the effect of substrate compositions on mycelial growth and secondary metabolites in Penicillium janthinellum and P declauxii (12). Among all of the carbon sources used, fructose was the most preferable sugar for ascochlorin production. Thus, yeast extract favored both biomass and ascochlorin production. Carbon and nitrogen sources are the most influential parameters on secondary metabolites of entomopathogenic fungi (13). One study with a Plackett-Burman design showed that sucrose, maltose, glucose and NaN[O.sub.3] were significant factors in zofimarin production (14). The use of the Plackett-Burman design and the central composite design for anhydromevalonolactone production has been reported, and the results suggested that sucrose, NaN[O.sub.3], yeast extract and [K.sub.2]HP[O.sub.4] were the key factors affecting anhydromevalonolactone production in a complex medium, whereas the major components required for a defined medium were NaN[O.sub.3], [K.sub.2]HP[O.sub.4], K[H.sub.2]P[O.sub.4] and trace elements, where a maximum anhydromevalonolactone production of 250 mg/L in the complex was obtained (15). Ascochlorin is a potent antiviral and antitumor antibiotic and is known to suppress breast cancer propagation (1, 7,8 9): the use of Microcera sp. BCC 17074 as a cell factory for ascochlorin production is of great interest for the production of a high amount of ascochlorin compared with other reported microorganisms. The cost of production medium in this study was approximately 0.5 $/L compared with the production in potato dextrose broth for 2.5 $/L. The production of ascochlorin by Microcera sp. BCC 17074 in this study has the potential for mass scale production, as the medium composition obtained from this study is very useful for enhancing its production. The high chloride-containing substrate might be corrosive to the stainless steel of the bioreactor, so perhaps feeding the chloride-containing substrate at the stationary phase of growth might be possible for process optimization of ascochlorin production. Furthermore, the major production of ascochlorin by Microcera sp. BCC 17074 is also beneficial for the downstream and purification processes, as it can avoid unwanted derivatives. This makes Microcera sp. BCC 17074 very attractive for ascochlorin production (7). The present study revealed that Microcera sp. BCC 17074 is a highly efficient cell factory for the ascochlorin production. The optimized medium conditions were successfully applied to incubation in a 5-L bioreactor, which demonstrated the feasibility of ascochlorin mass production.

ACKNOWLEDGEMENTS

We would like to thank Dr. Jean-Jacques Sanglier for the writing and editing of this manuscript as well as for consulting on the statistical analyses.

REFERENCES

(1.) Tamura, G., Suzuki, S., Takatsuki, A., Ando, K., Arima, K. Ascochlorin, a new antibiotic, found by paper-disc agar-diffusion method. I Isolation, biological and chemical properties of ascochlorin. J. Antibiot., 1968; 21: 539-544.

(2.) Sasaki, H., Hosokawa, T., Nawata, Y., Ando, K. Isolation and structure of ascochlorin and its analogs. Agric. Biol. Chem., 1974; 38: 1463-1466.

(3.) Aldridge, D.C., Borrow, A., Foster, R.G., Large, M.S., Spencer, H., Turner, W.B. Metabolites of Nectria coccinea. J. Chem. Soc. Perkin Trans., 1972; 1: 2136-2141.

(4.) Cagnoli-Bellavita, N., Ceccherelli, P., Fringuelli, R., Ribaldi, M. Ascochlorin: a terpenoid metabolite from Acremonium luzulae. Phytochem., 1975; 14: 807.

(5.) Seephonkai, P., Isaka, M., Kittakoop, P., Boonudomlap, U., Thebtaranonth, Y. A novel ascochlorin glycoside from the insect pathogenic fungus Verticillium hemipterigenum BCC 2370. J. Antibiot., 2004; 57: 10-16.

(6.) Kawaguchi, M., Fukuda, T., Uchida, R., Nonaka, K., Masuma, R., Tomoda, H. A new ascochlorin derivative from Cylindrocarpon sp. FKI-4602. J. Antibiot. 2013; 66: 23-29.

(7.) Isaka, M., Yangchum, A., Supothina, S., Laksanacharoen, P., Luangsa-ard, J.J., HywelJones, N.L. Ascochlorin derivatives from the leafhopper pathogenic fungus Microcera sp. BCC 17074. J. Antibiot, 2015; 68: 47-51.

(8.) Berry, E.A., Huang, L.S., Lee, D.W., Daldal, F., Nagai, K., Minagawa, N. Ascochlorin is a novel, specific inhibitor of the mitochondrial. Biochimica etBiophysica Acta., 2010; 1797: 360-370.

(9.) Takatsuki, A., Tamura, G., Arima, K. Antiviral and Antitumor Antibiotics. XIV. Effects of Ascochlorin and Other Respiration Inhibitors on Multiplication of Newcastle Disease Virus in Cultured Cells. Appl. Microbiol., 1969; 17: 825-829.

(10.) Nakajima, H., Mizuta, N., Sakaguchi, K., Fujiwara, I., Mizuta, M., Furukawa, C., Chang, Y.C., Magae, J. Aberrant Expression of Fra-1 in Estrogen Receptor-negative Breast Cancers and Suppression of their Propagation In Vivo by Ascochlorin, an Antibiotic that Inhibits Cellular Activator Protein-1 Activity. J. Antibiot., 2007; 60: 682-689.

(11.) Kang, J.H., Park, K.K., Lee, I.S., Magae, J., Ando, K., Kim, C.H., Chang, Y.C. Proteome analysis of responses to ascochlorin in a human osteosarcoma cell line by 2-D gel electrophoresis and MALDI-TOF MS. J. Proteome Res., 2006; 5: 2620-31.

(12.) Zain, M.E., El-Sheikh, H.H., Soliman, H.G., Khalil, A.M. Effect of certain chemical compounds on secondary metabolites of Penicillium janthinellum and P. duclauxii. J. Saudi Chem. Soc., 2011; 15: 239-246.

(13.) Prathumpai, W., Kocharin, K. Phomalactone Optimization and Production of Entomopathogenic Fungi; Ophiocordyceps communis BCC 1842 and BCC 2763. See comment in PubMed Commons belowPrep. Biochem. Biotechnol., 2014; 46: 44-48.

(14.) Chaichanan, J., W iyakrutta, S., Pongtharangkul, T., Isarangkul, D., Meevootisom, V. Optimization of zofimarin production by an endophytic fungus, Xylaria sp. Acra L38. Braz. J. Microbio, 2014; 45: 287-293.

(15.) Wattanachaisaereekul, S, Tachaleat, A., Punya, J., Haritakun, R., Boonlarppradab, C., Cheevadhanarak, S. Assessing medium constituents for optimal heterologous production of anhydromevalonolactone in recombinant Aspergillus oryzae. AMB Express., 2014; 4: 52.

Wai Prathumpai *, Pranee Rachtawee and Masahiko Isaka

Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Paholyothin Rd., Klong Nueng, Klong Luang, Pathum Thani, 12120, Thailand.

(Received: 13 January 2016; accepted: 19 April 2016)

* To whom all correspondence should be addressed.

Tel.: +66 2564 6700 ext. 3525-6, fax: +66 2564 6707; E-mail: wai.pra@biotec.or.th

Caption: Fig. 1. Ascochlorin structure

Caption: Fig. 2. Effects of ammonium chloride and sodium chloride on ascochlorin

Caption: Fig. 3. Time profile of biomass and ascochlorin production by Microcera sp. BCC 17074 in a 5-L bioreactor.
Table 1. Biomass and ascochlorin production by Microcera sp. BCC 17074
on different carbon and nitrogen sources using general factorial
design

Carbon            Nitrogen sources    Biomass
sources(20 g/L)   (4 g/L)             (g/L)

maltose           ammonium chloride   0.85 [+ or -] 0.02
trehalose         ammonium chloride   1.25 [+ or -] 0.04
glucose           ammonium chloride   1.0 [+ or -] 0.07
fructose          ammonium chloride   1.70 [+ or -] 0.01
galactose         ammonium chloride   0.8 [+ or -] 0.04
sorbitol          ammonium chloride   0.85 [+ or -] 0.04
maltose           malt extract        6.7 [+ or -] 0.07
trehalose         malt extract        1.65 [+ or -] 0.04
glucose           malt extract        1.65 [+ or -] 0.11
fructose          malt extract        1.75 [+ or -] 0.04
galactose         malt extract        1.8 [+ or -] 0.04
sorbitol          malt extract        2.35 [+ or -] 0.11
maltose           yeast extract       6.95 [+ or -] 0.04
trehalose         yeast extract       12.5 [+ or -] 0.21
glucose           yeast extract       10.9 [+ or -] 0.07
fructose          yeast extract       12.1 [+ or -] 0.64
galactose         yeast extract       8.2 [+ or -] 0.57
sorbitol          yeast extract       8.55 [+ or -] 0.32
maltose           meat extract        4.85 [+ or -] 0.11
trehalose         meat extract        12.1 [+ or -] 1.34
glucose           meat extract        9.9 [+ or -] 0.07
fructose          meat extract        12.6 [+ or -] 2.40 *
galactose         meat extract        10.55 [+ or -] 1.03
sorbitol          meat extract        7.35 [+ or -] 0.46
maltose           tryptone            7.55 [+ or -] 0.67
trehalose         tryptone            6.55 [+ or -] 0.32
glucose           tryptone            5.95 [+ or -] 0.04
fructose          tryptone            4.95 [+ or -] 0.04
galactose         tryptone            6.45 [+ or -] 0.39
sorbitol          tryptone            4.55 [+ or -] 0.32

Carbon            Ascochlorin             Ascochlorin
sources(20 g/L)   (mg/L)                  (mg/g DW)

maltose           0.56 [+ or -] 0.02      0.66 [+ or -] 0.02
trehalose         2.60 [+ or -] 0.04      2.08 [+ or -] 0.03 *
glucose           1.97 [+ or -] 0.05      1.97 [+ or -] 0.05
fructose          3.13 [+ or -] 0.05      1.84 [+ or -] 0.03
galactose         1.27 [+ or -] 0.14      1.59 [+ or -] 0.18
sorbitol          1.06 [+ or -] 0.02      1.25 [+ or -] 0.02
maltose           0.57 [+ or -] 0.04      0.09 [+ or -] 0.01
trehalose         1.02 [+ or -] 0.01      0.62 [+ or -] 0.01
glucose           2.55 [+ or -] 0.03      1.55 [+ or -] 0.02
fructose          1.04 [+ or -] 0.01      0.60 [+ or -] 0.01
galactose         2.32 [+ or -] 0.01      1.29 [+ or -] 0.01
sorbitol          3.39 [+ or -] 0.01      1.44 [+ or -] 0.01
maltose           1.92 [+ or -] 0.01      0.28 [+ or -] 0.01
trehalose         14.64 [+ or -] 0.01     1.17 [+ or -] 0.01
glucose           6.63 [+ or -] 0.01      0.61 [+ or -] 0.01
fructose          20.19 [+ or -] 0.01 *   1.67 [+ or -] 0.01
galactose         7.71 [+ or -] 0.02      0.94 [+ or -] 0.01
sorbitol          2.69 [+ or -] 0.01      0.32 [+ or -] 0.01
maltose           0.08 [+ or -] 0.01      0.02 [+ or -] 0.01
trehalose         13.26 [+ or -] 0.01     1.10 [+ or -] 0.01
glucose           12.14 [+ or -] 0.01     0.96 [+ or -] 0.01
fructose          11.24 [+ or -] 0.01     0.89 [+ or -] 0.01
galactose         5.83 [+ or -] 0.01      0.55 [+ or -] 0.01
sorbitol          4.77 [+ or -] 0.01      0.65 [+ or -] 0.01
maltose           6.88 [+ or -] 0.01      0.91 [+ or -] 0.01
trehalose         2.98 [+ or -] 0.01      0.46 [+ or -] 0.01
glucose           1.35 [+ or -] 0.01      0.23 [+ or -] 0.01
fructose          2.71 [+ or -] 0.01      0.55 [+ or -] 0.01
galactose         2.50 [+ or -] 0.01      0.39 [+ or -] 0.01
sorbitol          0.13 [+ or -] 0.01      0.03 [+ or -] 0.01

* Maximum values

Table 2. Analysis of variance (ANOVA) for the general fractional
factorial design of biomass production by Microcera sp. BCC 17074

Source                    Sum of squares   D.F.   Mean square

Model * A-Carbon source    460.4316.15     106     46.042.69
B-Nitrogen source             444.27        4       111.07
Residual                      83.15         24       3.46
Corrected total               543.58        34

Source                     F-value    Probability (P) > F

Model * A-Carbon source   13.290.78     < 0.00010.5958
B-Nitrogen source           32.06          < 0.0001
Residual
Corrected total

[R.sup.2] = 0.8470, adj-[R.sup.2] = 0.7833, SD = 1.86, Mean = 5.89,
%CV = 31.59

* significant

Table 3. Analysis of variance (ANOVA) for the general fractional
factorial design of ascochlorin production by Microcera sp. BCC 17074

Source                    Sum of squares    D.F.       Mean square

Model * A-Carbon source   2.123E61.675E5     106     2.123E527912.33
B-Nitrogen source            1.956E6          4          4.889E5
Residual                                   6.033E5         24
Corrected total              2.726E6         34

Source                    F-value    Probability (P) > F

Model * A-Carbon source   8.451.11     < 0.00010.3855
B-Nitrogen source          19.45          < 0.0001
Residual                  25139.30
Corrected total

[R.sup.2] = 0.7787, adj-[R.sup.2] = 0.6865, SD = 158.55, Mean =
143.64, %CV = 110.38

Table 4. Biomass and ascochlorin production by Microcera sp. BCC 17074
using a Plackett-Burman design with 9 factors ([2.sup.n-1]) and 2
dummies

STD                            Factors

      Fructose   (g/L) Yeast   Ammonium   NaCl    Ca[Cl.sub.2].2
       (g/L)       extract     chloride   (g/L)     [H.sub.2]0
                    (g/L)       (g/L)                 (g/L)

1        60           6           0         2          0.2
2        20           6           4         0          0.2
3        60           2           4         2           0
4        20           6           0         2          0.2
5        20           2           4         0          0.2
6        20           2           0         2           0
7        60           2           0         0          0.2
8        60           6           0         0           0
9        60           6           4         0           0
10       20           6           4         2           0
11       60           2           4         2          0.2
12       20           2           0         0           0
13       40           4           2         1          0.1
14       40           4           2         1          0.1
15       40           4           2         1          0.1
16       20           2           4         0           0
17       60           2           0         2           0
18       20           6           0         0          0.2
19       60           2           4         0           0
20       60           6           0         2           0
21       60           6           4         0          0.2
22       20           6           4         2           0
23       20           2           4         2          0.2
24       20           2           0         2          0.2
25       60           2           0         0          0.2
26       20           6           0         0           0
27       60           6           4         2          0.2
28       40           4           2         1          0.1

STD                      Factors

      K[H.sub.2]   [K.sub.2]HP[O.sub.4]    KC1     Trace
      P[O.sub.4]          (g/L)           (g/L)   elements
        (g/L)                                      (mL/L)

1         2                 0               0        0
2         2                 2               0        0
3         2                 2               2        0
4         0                 2               2        2
5         2                 0               2        2
6         2                 2               0        2
7         0                 2               2        0
8         2                 0               2        2
9         0                 2               0        2
10        0                 0               2        0
11        0                 0               0        2
12        0                 0               0        0
13        1                 1               1        1
14        1                 1               1        1
15        1                 1               1        1
16        0                 2               2        2
17        0                 0               2        2
18        0                 0               0        2
19        2                 0               0        0
20        0                 2               0        0
21        0                 0               2        0
22        2                 0               0        2
23        0                 2               0        0
24        2                 0               2        0
25        2                 2               0        2
26        2                 2               2        0
27        2                 2               2        2
28        1                 1               1        1

STD          Biomass               Ascochlorin

                                     (mg/L)

1      15.75 [+ or -] 0.21    24.60 [+ or -] 0.76 *
2      7.95 [+ or -] 0.86      2.40 [+ or -] 0.09
3      13.05 [+ or -] 0.64     19.43 [+ or -] 0.74
4      8.90 [+ or -] 0.28      8.41 [+ or -] 0.75
5      8.25 [+ or -] 0.07      11.24 [+ or -] 0.15
6      3.60 [+ or -] 0.14      0.94 [+ or -] 0.02
7      7.75 [+ or -] 0.78      0.27 [+ or -] 0.02
8      10.80 [+ or -] 0.71     2.36 [+ or -] 0.30
9      12.00 [+ or -] 1.41     22.26 [+ or -] 2.89
10     11.00 [+ or -] 3.39     3.15 [+ or -] 0.11
11     9.50 [+ or -] 1.27      12.44 [+ or -] 0.56
12     6.20 [+ or -] 0.57      0.24 [+ or -] 0.04
13     12.05 [+ or -] 0.50     10.07 [+ or -] 0.16
14     11.50 [+ or -] 1.84     5.83 [+ or -] 0.04
15     11.75 [+ or -] 0.07     12.12 [+ or -] 0.27
16     8.90 [+ or -] 0.71      3.72 [+ or -] 0.23
17     7.00 [+ or -] 2.83      3.15 [+ or -] 0.06
18     9.70 [+ or -] 0.14               0
19     12.75 [+ or -] 1.91     10.87 [+ or -] 0.12
20    23.40 [+ or -] 1.84 *             0
21     16.15 [+ or -] 0.78     6.16 [+ or -] 0.22
22     17.15 [+ or -] 9.69     4.24 [+ or -] 0.07
23     8.70 [+ or -] 0.71      1.81 [+ or -] 0.02
24     6.25 [+ or -] 2.19               0
25     6.65 [+ or -] 0.07      1.71 [+ or -] 0.09
26     9.90 [+ or -] 1.70      3.95 [+ or -] 0.12
27     14.55 [+ or -] 1.49     4.24 [+ or -] 0.02
28     11.75 [+ or -] 2.10     13.31 [+ or -] 5.05

STD       Ascochlorin

            (mg/gDW)

1      1.56 [+ or -] 0.40
2      0.30 [+ or -] 0.16
3      1.49 [+ or -] 0.54
4      0.95 [+ or -] 0.08
5      1.36 [+ or -] 0.29
6      0.26 [+ or -] 0.20
7      0.04 [+ or -] 0.01
8      0.22 [+ or -] 0.03
9     1,86 [+ or -] 0.22 *
10     0.29 [+ or -] 0.01
11     1.31 [+ or -] 0.06
12     0.04 [+ or -] 0.01
13     0.84 [+ or -] 0.01
14     0.51 [+ or -] 0.01
15     1.03 [+ or -] 0.02
16     0.42 [+ or -] 0.02
17     0.45 [+ or -] 0.01
18             0
19    0.858 [+ or -] 0.01
20             0
21     0.38 [+ or -] 0.01
22     0.25 [+ or -] 0.01
23     0.02 [+ or -] 0.01
24             0
25     0.26 [+ or -] 0.01
26     0.40 [+ or -] 0.01
27     0.29 [+ or -] 0.01
28     1.13 [+ or -] 0.36

* Maximum values

Table 5. Analysis of variance (ANOVA) for a Plackett-Burman
design of biomass production by Microcera sp. BCC 17074

Source                       Sum of squares   D.F.   Mean square

Model *                          411.85        22       18.72
A-Fructose                       76.51         1        76.51
B-Yeast extract                  143.33        1       143.33
C-Ammonium chloride              24.10         1        24.10
D-Sodium chloride                19.89         1        19.89
E-Ca[Cl.sub.2].2[H.sub.2]O       10.21         1        10.21
F- K[H.sub.2]P[O.sub.4]           0.27         1        0.27
g-[K.sub.2]HP[O.sub.4]            1.11         1        1.11
H-KCl                             4.91         1        4.91
J-Trace elements                 19.89         1        19.89
Curvature                         5.79         1        5.79
Residual                         14.82         5        2.96
Lack of fit **                    5.87         1        5.87
Pure error **                     8.95         4        2.24
Corrected total                  455.51        29

Source                       F-value   Probability (P) > F

Model *                       6.32           0.0249
A-Fructose                    25.81          0.0038
B-Yeast extract               48.35          0.0009
C-Ammonium chloride           8.13           0.0358
D-Sodium chloride             6.71           0.0488
E-Ca[Cl.sub.2].2[H.sub.2]O    3.44           0.1227
F- K[H.sub.2]P[O.sub.4]       0.091          0.7746
g-[K.sub.2]HP[O.sub.4]        0.37           0.5682
H-KCl                         1.65           0.2546
J-Trace elements              6.71           0.0488
Curvature                     1.95           0.2212
Residual
Lack of fit **                2.63           0.1804
Pure error **
Corrected total

[R.sup.2] = 0.9653, adj-[R.sup.2] = 0.8124, SD = 1.72, Mean = 10.88,
%CV = 15.82

* significant, ** not significant

Table 6. Analysis of variance (ANOVA) for a Plackett-Burman
design of ascochlorin production by Microcera sp. BCC 17074.

Source                       Sum of squares   D.F.   Mean square

Model **                         990.0         22       45.0
A-Fructose                       189.20        1       189.20
B-Yeast extract                  10.61         1        10.61
C-Ammonium chloride              132.19        1       132.19
D-Sodium chloride                12.38         1        12.38
E-Ca[Cl.sub.2].2[H.sub.2]O       0.045         1        0.045
F-K[H.sub.2]P[O.sub.4]           24.79         1        24.79
g-[K.sub.2]HP[O.sub.4]            3.60         1        3.60
H-KCl                             9.91         1        9.91
J-Trace elements                  0.14         1        0.14
Curvature                        44.13         1        44.13
Residual                         105.93        5        21.19
Lack of fit **                   34.26         1        34.26
Pure error **                    71.67         4        17.92
Corrected total                 1297.98        29

Source                       F-value   Probability (P) > F

Model **                      2.12           0.2061
A-Fructose                    8.93           0.0305
B-Yeast extract               0.50           0.5107
C-Ammonium chloride           6.24           0.0546
D-Sodium chloride             0.58           0.4791
E-Ca[Cl.sub.2].2[H.sub.2]O   2.12E-3         0.9650
F-K[H.sub.2]P[O.sub.4]        1.17           0.3288
g-[K.sub.2]HP[O.sub.4]        0.17           0.6973
H-KCl                         0.47           0.5245
J-Trace elements             6.59E-3         0.9385
Curvature                     2.08           0.2085
Residual
Lack of fit **                1.91           0.2389
Pure error **
Corrected total

[R.sup.2] = 0.9033, adj-[R.sup.2] = 0.4781, SD = 4.60, Mean = 6.76,
%CV = 68.14

** not significant

Table 7. Biomass and ascochlorin production by Microcera sp. BCC 17074
using a central composite design

STD                        Factors
      Fructose    Yeast    Ammonium   NaCl    K[H.sub.2]P[O.sub.4]
       (g/L)     extract   chloride   (g/L)          (g/L)
                  (g/L)     (g/L)

1        60         6         2         4              2
2        60         2         4         4              2
3        20         6         4         2              4
4        60         6         4         2              2
5        60         6         2         2              4
6        60         2         2         4              4
7        20         2         4         4              4
8        20         6         2         4              4
9        60         2         4         2              4
10       20         6         4         4              2
11       20         2         2         2              2
12     10.09        4         3         3              3
13     69.91        4         3         3              3
14       40       1.01        3         3              3
15       40       6.99        3         3              3
16       40         4        1.5        3              3
17       40         4        4.5        3              3
18       40         4         3        1.5             3
19       40         4         3        4.5             3
20       40         4         3         3             1.5
21       40         4         3         3             4.5
22       40         4         3         3              3

STD         Biomass               Ascochlorin
             (g/L)                   (mg/L)

1      19.4 [+ or -] 0.42     13.12 [+ or -] 0.62
2     12.55 [+ or -] 1.03      7.55 [+ or -] 0.40
3      10.7 [+ or -] 0.92      1.48 [+ or -] 0.02
4     19.9 [+ or -] 0.78 *    21.74 [+ or -] 0.18
5      18.3 [+ or -] 0.49     14.22 [+ or -] 0.55
6     14.95 [+ or -] 0.74      1.21 [+ or -] 0.07
7      8.1 [+ or -] 0.64       1.75 [+ or -] 0.04
8      12.5 [+ or -] 0.35      2.38 [+ or -] 0.09
9     12.25 [+ or -] 1.24      6.14 [+ or -] 0.26
10    11.25 [+ or -] 0.53      1.86 [+ or -] 0.03
11     6.2 [+ or -] 0.57       1.22 [+ or -] 0.06
12     2.9 [+ or -] 0.78       0.03 [+ or -] 0.01
13     4.3 [+ or -] 0.49     0.09 [+ or -] 1.950.01
14     7.5 [+ or -] 1.06       5.45 [+ or -] 0.39
15     15.8 [+ or -] 1.56     14.20 [+ or -] 0.57
16     15.0 [+ or -] 0.71     14.97 [+ or -] 0.02
17     14.5 [+ or -] 1.06     17.78 [+ or -] 0.16
18    16.25 [+ or -] 1.24      0.80 [+ or -] 0.14
19     17.2 [+ or -] 1.56     25.60 [+ or -] 0.28
20     14.4 [+ or -] 1.70     29.20 [+ or -] 0.57
21     18.8 [+ or -] 1.14    41.12 [+ or -] 0.62 *
22    14.12 [+ or -] 0.65     15.46 [+ or -] 0.05

STD       Ascochlorin
           (mg/g DW)

1      0.68 [+ or -] 0.03
2      0.59 [+ or -] 0.03
3      0.14 [+ or -] 0.01
4      1.09 [+ or -] 0.01
5      0.78 [+ or -] 0.03
6      0.08 [+ or -] 0.01
7      0.22 [+ or -] 0.01
8      0.19 [+ or -] 0.01
9      0.50 [+ or -] 0.02
10     0.17 [+ or -] 0.01
11     0.20 [+ or -] 0.01
12     0.01 [+ or -] 0.00
13     0.02 [+ or -] 0.00
14     0.73 [+ or -] 0.05
15     0.90 [+ or -] 0.04
16     1.00 [+ or -] 0.01
17     1.23 [+ or -] 0.01
18     0.05 [+ or -] 0.01
19     1.49 [+ or -] .02
20     2.03 [+ or -] 0.04
21    2.19 [+ or -] 0.03 *
22     1.10 [+ or -] 0.01

* Maximum values

Table 8. Analysis of variance (ANOVA) for a central composite
design of biomass production by Microcera sp. BCC 17074

Source                   Sum of squares   D.F.   Mean square   F-value

Model *                      486.27        20       24.31       18.95
A-Fructose                    0.98         1        0.98        0.76
B-Yeast extract              34.44         1        34.44       26.85
C-Ammonium chloride           0.12         1        0.12        0.097
D-Sodium chloride             0.45         1        0.45        0.35
F-K[H.sub.2]P[O.sub.4]        9.68         1        9.68        7.55
AB                           14.19         1       114.19       11.06
AC                           11.09         1        11.09       8.64
AD                           11.77         1        11.77       9.17
AF                            1.22         1        1.22        0.95
BC                            1.79         1        1.79        1.40
BD                            2.52         1        2.52        1.96
BF                           16.06         1        16.06       12.52
CD                            1.92         1        1.92        1.50
CF                           12.23         1        12.23       9.53
DF                            6.29         1        6.29        4.90
[A.sup.2]                    191.57        1       191.57      149.33
[B.sup.2]                     4.31         1        4.31        3.36
[C.sup.2]                     6.02         1        6.02        4.70
[D.sup.2]                    28.52         1        28.52       22.23
[E.sup.2]                    26.61         1        26.61       20.74
Residual                      3.85         3        1.28
Lack of fit **                3.0          1         3.0        7.04
Pure error                    0.85         2        0.43
Corrected total              490.11        23

Source                    Probability (P) > F

Model *                         0.0165
A-Fructose                      0.4464
B-Yeast extract                 0.0140
C-Ammonium chloride             0.7754
D-Sodium chloride               0.5949
F-K[H.sub.2]P[O.sub.4]          0.0709
AB                              0.0449
AC                              0.0605
AD                              0.0564
AF                              0.4010
BC                              0.3223
BD                              0.2559
BF                              0.0384
CD                              0.3086
CF                              0.0538
DF                              0.1137
[A.sup.2]                       0.0012
[B.sup.2]                       0.1642
[C.sup.2]                       0.1188
[D.sup.2]                       0.0181
[E.sup.2]                       0.0198
Residual
Lack of fit **                  0.1176
Pure error
Corrected total

[R.sup.2] = 0.99921, adj-[R.sup.2] = 0.9398, SD = 1.13, Mean = 13.13,
%CV = 8.63

* significant, ** not significant

Table 9. Analysis of variance (ANOVA) for a central composite
design of ascochlorin production by Microcera sp. BCC 17074

Source                Sum of squares   D.F.   Mean square   F-value

Model *                  2589.28        20      129.46       2.69
A-Fructose               1.49E-3        1       1.49E-3     3.10E-5
B-Yeast extract           38.22         1        38.22       0.79
C-Ammonium chloride        3.95         1        3.95        0.082
D-Sodium chloride         307.48        1       307.48       6.39
E-K[H.sub.2]P[O.sub.4]    71.09         1        71.09       1.48
AB                        322.54        1       322.54       6.71
AC                        246.82        1       246.82       5.13
AD                         0.80         1        0.80        0.017
AE                        32.27         1        32.27       0.67
BC                        52.72         1        52.72       1.10
BD                        50.31         1        50.31       1.05
BE                        0.025         1        0.025      5.30E-4
CD                        41.77         1        41.77       0.87
CE                         0.21         1        0.21       4.37E-3
DE                        179.17        1       179.17       3.73
[A.sup.2]                 859.49        1       859.49       17.87
[B.sup.2]                 226.46        1       226.46       4.71
[C.sup.2]                 30.04         1        30.04       0.62
[D.sup.2]                 102.22        1       102.22       2.13
[E.sup.2]                 482.95        1       482.95       10.04
Residual                  144.29        3        48.10
Lack of fit **            59.53         1        59.53       1.40
Pure error                84.76         2        42.38
Corrected total          2733.57        23

Source                Probability (P) > F

Model *                     0.0112
A-Fructose                  0.9959
B-Yeast extract             0.4384
C-Ammonium chloride         0.7931
D-Sodium chloride           0.0855
E-K[H.sub.2]P[O.sub.4]      0.3110
AB                          0.0811
AC                          0.1084
AD                          0.9053
AE                          0.4727
BC                          0.3720
BD                          0.3817
BE                          0.9831
CD                          0.4201
CE                          0.9514
DE                          0.1491
[A.sup.2]                   0.0242
[B.sup.2]                   0.1185
[C.sup.2]                   0.4870
[D.sup.2]                   0.2409
[E.sup.2]                   0.0505
Residual
Lack of fit **              0.3577
Pure error
Corrected total

[R.sup.2] = 0.9472, adj-[R.sup.2] = 0.5953, SD = 6.94, Mean = 11.17,
%CV = 62.07

* significant, ** not significant
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Author:Prathumpai, Wai; Rachtawee, Pranee; Isaka, Masahiko
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Sep 1, 2016
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