Exploring untapped bacterial communities and potential polypropylene-degrading enzymes from mangrove sediment through metagenomics analysis.

The versatility of plastic has resulted in huge amounts being consumed annually. Mismanagement of post-consumption plastic material has led to plastic waste pollution. Biodegradation of plastic by microorganisms has emerged as a potential solution to this problem. Therefore, this study aimed to investigate the microbial communities involved in the biodegradation of polypropylene (PP). Mangrove soil was enriched with virgin PP sheets or chemically pretreated PP comparing between 2 and 4 months enrichment to promote the growth of bacteria involved in PP biodegradation. The diversity of the resulting microbial communities was accessed through 16S metagenomic sequencing. The results indicated that Xanthomonadaceae, unclassified Gaiellales, and Nocardioidaceae were promoted during the enrichment. Additionally, shotgun metagenomics was used to investigate enzymes involved in plastic biodegradation. The results revealed the presence of various putative plastic-degrading enzymes in the mangrove soil, including alcohol dehydrogenase, aldehyde dehydrogenase, and alkane hydroxylase. The degradation of PP plastic was determined using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM), and Water Contact Angle measurements. The FTIR spectra showed a reduced peak intensity of enriched and pretreated PP compared to the control. SEM images revealed the presence of bacterial biofilms as well as cracks on the PP surface. Corresponding to the FTIR and SEM analysis, the water contact angle measurement indicated a decrease in the hydrophobicity of PP and pretreated PP surface during the enrichment.

Naturally occurring quercetin and myricetin as potent inhibitors for human ectonucleotide pyrophosphatase/phosphodiesterase 1.

Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) is a key enzyme in purinergic signaling pathways responsible for cell-to-cell communications and regulation of several fundamental pathophysiological processes. In this study, Kyoto Green, a rapid chemical sensor of pyrophosphate, was employed to screen for effective ENPP1 inhibitors among five representative flavonoids (quercetin, myricetin, morin, kaempferol, and quercetin-3-glucoside), five nucleosides (adenosine, guanosine, inosine, uridine, and cytidine), and five deoxynucleosides (2'- and 3'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyinosine, and 2'-deoxyuridine). Conventional colorimetric, fluorescence, and bioluminescence assays revealed that ENPP1 was effectively inhibited by quercetin (Ki ~ 4 nM) and myricetin (Ki ~ 32 nM) when ATP was used as a substrate at pH 7.4. In silico analysis indicated that the presence of a chromone scaffold, particularly one containing a hydroxyl group at the 3' position on the B ring, may promote binding to the active site pocket of ENPP1 and enhance inhibition. This study demonstrated that the naturally derived quercetin and myricetin could effectively inhibit ENPP1 enzymatic activity and may offer health benefits in arthritis management.

A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways.

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.

The Effect of Different pH Conditions on Peptides' Separation from the Skipjack Dark Meat Hydrolysate Using Ceramic Ultrafiltration.

The conversion of Skipjack (Katsuwonus pelamis) dark meat into a hydrolysate via enzymatic hydrolysis is a promising approach to increase the value of tuna by-products as a source of bioactive peptides. Skipjack dark meat hydrolysate (SDMH) contains various sizes and sequences of peptides. To obtain and concentrate the targeted small peptides from SDMH, ultrafiltration, a key unit operation process, was employed to fractionate the protein hydrolysate due to its simplicity and productivity. The objective of this study was to investigate the effect of the feed pH on the membrane performance based on the permeate flux and the transmission of peptides. The fractionation of SDMH was performed using a ceramic membrane (molecular weight cut-off of 1 kDa) with three different pH values (5, 7, and 9) at various transmembrane pressures (TMP) (2.85, 3.85, and 4.85 bar). A high permeate flux and transmission were obtained at pH 9 due to the repulsive interactions between peptides and the membrane surface, leading to the reduction in concentration polarization that could promote high transmission. In addition, the combination of low TMP (2.85 bar) and pH 9 helped to even minimize the fouling formation tendency, providing the highest peptide transmission in this study. The fractionation process resulted in the enhancement of small peptides (MW < 0.3 kDa). The amino acid profiles were different at each pH, affirming the charge effect from the pH changes. In conclusion, the performance of the membrane was affected by the pH of the hydrolysate. Additionally, the ultrafiltration method served as an alternate method of peptide separation on a commercial scale.

Kinetic model of a newly-isolated Lysinibacillus sp. strain YL and elastic properties of its biogenic CaCO3 towards biocement application.

BACKGROUND: Biocement, calcifying bacteria-incorporated cement, offers an environmentally-friendly way to increase the cement lifespan. This work aimed to investigate the potential use of Lysinibacillus sp. strain YL towards biocement application in both theoretical and experimental ways. METHODS AND RESULTS: Strain YL was grown using calcium acetate (Ca(C2 H3 O2 )2 ), calcium chloride (CaCl2 ) and calcium nitrate (Ca(NO3 )2 ). Maximum bacterial growth of ~0.09 hr-1 and the highest amount of CaCO3 precipitation of ~8.0 g/L were obtained when using Ca(C2 H3 O2 )2 . The SEM and XRD results confirmed that biogenic CaCO3 were calcites. The bulk, Young's and shear moduli of biogenic CaCO3 calculated via the VRH approximation were ~1.5-2.3 times larger than those of ordinary Portland cement. The Poisson's ratio was 0.382 and negative in some directions, suggesting its ductility and auxetic behaviors. The new model was developed to explain the growth kinetic of strain YL in the presence of Ca(C2 H3 O2 )2 , whose concentration was optimized for biocement experiments. Strain YL could increase the compressive strength of cement up to ~50% higher than that of the uninoculated cement. CONCLUSION: Strain YL is a promising candidate for biocement applications. This work represents the trials of experiments and models allowing quantitatively comparison with large-scale production in the future.

A fixed-film bioscrubber of Microbacterium esteraromaticum SBS1-7 for toluene/styrene biodegradation.

In the present study, a fixed-film bioscrubber (FFBS) of BTEX-degrading bacterium Microbacterium esteraromaticum SBS1-7 with 'AQUAPOROUSGEL® or APG' supporting material was continuously fed with toluene- or styrene-contaminated gas stream for 172 days. Response Surface Methodology (RSM) was used to optimize the biofilm formation on APG as well as the toluene biodegradation in mineral salt medium (MM). The results suggested that 1000 ppm of yeast extract (YE) was necessary for biofilm formation of SBS1-7. The optimized combination of YE and toluene concentration exhibiting the highest biofilm formation and toluene removal was further employed in an up-scale FFBS operation. The maximum Elimination Capacity (ECmax) of 203 g·m-3·h-1 was obtained at the toluene Inlet Loading Rate (ILR) of 295 g·m-3·h-1. FFBS of SBS1-7 was able to withstand a 5-day shutdown and required only 24 h to recover. Moreover, when the inlet Volatile Organic Compound was shifted to styrene, FFBS required only 24 h for adaptation and the system was able to efficiently remove ~95% of styrene after that. Finally, the performance of the bioscrubber when operated in 2 different modes of operation (FFBS vs Biotricking Filter or BTF) were compared. This study evidently demonstrated the robustness and stability of FFBS with M. esteraromaticum SBS1-7.

BTEX biodegradation by Bacillus amyloliquefaciens subsp. plantarum W1 and its proposed BTEX biodegradation pathways.

Benzene, toluene, ethylbenzene and (p-, m- and o-) xylene (BTEX) are classified as main pollutants by several environmental protection agencies. In this study, a non-pathogenic, Gram-positive rod-shape bacterium with an ability to degrade all six BTEX compounds, employed as an individual substrate or as a mixture, was isolated. The bacterial isolate was identified as Bacillus amyloliquefaciens subsp. plantarum strain W1. An overall BTEX biodegradation (as individual substrates) by strain W1 could be ranked as: toluene > benzene, ethylbenzene, p-xylene > m-xylene > o-xylene. When presented in a BTEX mixture, m-xylene and o-xylene biodegradation was slightly improved suggesting an induction effect by other BTEX components. BTEX biodegradation pathways of strain W1 were proposed based on analyses of its metabolic intermediates identified by LC-MS/MS. Detected activity of several putative monooxygenases and dioxygenases suggested the versatility of strain W1. Thus far, this is the first report of biodegradation pathways for all of the six BTEX compounds by a unique bacterium of the genus Bacillus. Moreover, B. amyloliquefaciens subsp. plantarum W1 could be a good candidate for an in situ bioremediation considering its Generally Recognized as Safe (GRAS) status and a possibility to serve as a plant growth-promoting rhizobacterium (PGPR).

Hydroxypropyl methylcellulose enhances the stability of o/w Pickering emulsions stabilized with chitosan and the whole cells of Lactococcus lactis IO-1.

Hydroxypropyl methylcellulose (HPMC) was used as a co-emulsifier with chitosan and the whole cells of bacteriocin-producing Lactococcus lactis IO-1 (L. lactis IO-1) for the preparation of bacteria interface Pickering emulsion. The obtained emulsion exhibited a high stability against centrifugation force, ionic strength and low temperature, with the whole cells of L. lactis IO-1 located at the oil/water interface. Because L.lactis IO-1 was found to produce peptide lantibiotic against several strains of Gram-positive food pathogen, the highly stable emulsion demonstrated in this study exhibited high potential as an antimicrobial emulsion with several health benefits of chitosan and HPLC useful for food industry.

Engineering of Corynebacterium glutamicum as a prototrophic pyruvate-producing strain: Characterization of a ramA-deficient mutant and its application for metabolic engineering.

To construct a prototrophic Corynebacterium glutamicum strain that efficiently produces pyruvate from glucose, the effects of inactivating RamA, a global regulator responsible for activating the oxidative tricarboxylic acid (TCA) cycle, on glucose metabolism were investigated. ΔramA showed an increased specific glucose consumption rate, decreased growth, comparable pyruvate production, higher formation of lactate and acetate, and lower accumulation of succinate and 2-oxoglutarate compared to the wild type. A significant decrease in pyruvate dehydrogenase complex activity was observed for ΔramA, indicating reduced carbon flow to the TCA cycle in ΔramA. To create an efficient pyruvate producer, the ramA gene was deleted in a strain lacking the genes involved in all known lactate- and acetate-producing pathways. The resulting mutant produced 161 mM pyruvate from 222 mM glucose, which was significantly higher than that of the parent (89.3 mM; 1.80-fold).

BTEX- and naphthalene-degrading bacterium Microbacterium esteraromaticum strain SBS1-7 isolated from estuarine sediment.

In this study, a non-pathogenic, BTEX-degrading Microbacterium esteraromaticum SBS1-7 was isolated from estuarine sediment in Thailand via an enrichment technique. M. esteraromaticum SBS1-7 was able to degrade all six BTEX components, in both liquid medium and soil slurry system, when BTEX was supplied as an individual component or a mixture. It exhibited a high level of tolerance towards a wide range of hydrocarbons and also utilized alkanes and naphthalene. Detection of metabolites produced during BTEX and naphthalene degradation revealed highly extensive biodegradation pathways used by M. esteraromaticum SBS1-7. Toluene was metabolized via activities of both monooxygenase (toluene 4-monooxygenase or T4MO) and dioxygenases (toluene dioxygenase or TDO and naphthalene 1,2-dioxygenase or NDO). Benzene was metabolized via phenol, possibly by an activity of T4MO. Ethylbenzene was converted into styrene and 1-phenethyl alcohol by a well-documented activity of NDO. Dioxidation of ethylbenzene, possibly by ethylbenzene dioxygenase or EBDO, was also found. All xylene isomers were converted into their corresponding alcohols via an activity of NDO while naphthalene was metabolized via dioxidation reaction by the same enzyme. This study is, by far, the first direct evidence of BTEX biodegradation by a non-pathogenic, rhizosphere bacterium M. esteraromaticum.

Production of 1,3-diols in Escherichia coli.

To expand the diversity of chemical compounds produced through microbial conversion, a platform pathway for the production of widely used industrial chemicals, 1,3-diols, was engineered in Escherichia coli. The pathway was designed by modifying the previously reported (R)-1,3-butanediol synthetic pathway to consist of pct (propionate CoA-transferase) from Megasphaera elsdenii, bktB (thiolase), phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, bld (butyraldehyde dehydrogenase) from Clostridium saccharoperbutylacetonicum, and the endogenous alcohol dehydrogenase(s) of E. coli. The recombinant E. coli strains produced 1,3-pentanediol, 4-methyl-1,3-pentanediol, and 1,2,4-butanetriol, together with 1,3-butanediol, from mixtures of glucose and propionate, isobutyrate, and glycolate, respectively, in shake flask cultures. To the best of our knowledge, this is the first report of microbial production of 1,3-pentanediol and 4-methyl-1,3-pentanediol.

Turning hydrophilic bacteria into biorenewable hydrophobic material with potential antimicrobial activity via interaction with chitosan.

Alteration of a bacteriocin-producing hydrophilic bacterium, Lactococcus lactis IO-1, into a hydrophobic material with potential antimicrobial activity using chitosan was investigated and compared with five other bacterial species with industrial importance. The negatively charged bacterial cells were neutralized by positively charged chitosan, resulting in a significant increase in the hydrophobicity of the bacterial cell surface. The largest Gram-positive B. megaterium ATCC 14581 showed a moderate response to chitosan while the smaller E. coli DH5α, L. lactis IO-1 and P. putida F1 exhibited a significant response to an increase in chitosan concentration. Because L. lactis IO-1 is a good source for natural peptide lantibiotic that is highly effective against several strains of food spoilage organisms and pathogens, hydrophobic material derived from L. lactis IO-1 and chitosan is a promising novel material with antimicrobial activity for the food and pharmaceutical industries.

Butyrate production under aerobic growth conditions by engineered Escherichia coli.

Butyrate is an important industrial platform chemical. Although several groups have reported butyrate production under oxygen-limited conditions by a native producer, Clostridium tyrobutylicum, and by a metabolically engineered Escherichia coli, efforts to produce butyrate under aerobic growth conditions have met limited success. Here, we constructed a novel butyrate synthetic pathway that functions under aerobic growth conditions in E. coli, by modifying the 1-butanol synthetic pathway reported previously. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, and endogenous thioesterase(s) of E. coli. To evaluate the potential of this pathway for butyrate production, culture conditions, including pH, oxygen supply, and concentration of inorganic nitrogen sources, were optimized in a mini-jar fermentor. Under the optimal conditions, butyrate was produced at a concentration of up to 140 mM (12.3 g/L in terms of butyric acid) after 54 h of fed-batch culture.

Process optimization for an industrial-scale production of Diphtheria toxin by Corynebacterium diphtheriae PW8.

In this study, several parameters affecting the toxin production of Corynebacterium diphtheriae Parke Williams 8 (PW8) were investigated in detail. The comparison studies of amino acid profile in NZ Amine A-based medium (NZ medium) and beef digest-based medium (BD medium) suggested that an insufficient supply of amino acids was not responsible for low toxin yield observed in NZ medium. Supplementation of additional amino acids and growth promoting nutrient (in a form of yeast extract) into NZ medium enhanced only cell growth but not toxin production. Thus, BD medium was selected as the most suitable base medium for toxin production as it gave a significantly higher limit of flocculation (93 ± 0 Lf/ml) than NZ medium (46 ± 0 Lf/ml). Interestingly, a supplementation of 0.2% YE into BD medium resulted in a significant increase in growth as well as toxin production (235 ± 5 Lf/ml). In conclusion, consistently high toxin titer (174-239 Lf/ml) could be obtained from BD medium at a 5 L-scale production as long as 1) the protein content of BD medium was at least 24 g/L, 2) the iron content was below 0.15 ppm and 3) 0.2% YE was supplemented into the medium.

Kinetic properties and stability of glucose dehydrogenase from Bacillus amyloliquefaciens SB5 and its potential for cofactor regeneration.

Glucose dehydrogenases (GluDH) from Bacillus species offer several advantages over other NAD(P)H regeneration systems including high stability, inexpensive substrate, thermodynamically favorable reaction and flexibility to regenerate both NADH and NADPH. In this research, characteristics of GluDH from Bacillus amyloliquefaciens SB5 (GluDH-BA) was reported for the first time. Despite a highly similar amino acid sequence when comparing with GluDH from Bacillus subtilis (GluDH-BS), GluDH-BA exhibited significantly higher specific activity (4.7-fold) and stability when pH was higher than 6. While an optimum activity of GluDH-BA was observed at a temperature of 50 °C, the enzyme was stable only up to 42 °C. GluDH-BA exhibited an extreme tolerance towards n-hexane and its respective alcohols. The productivity of GluDH obtained in this study (8.42 mg-GluDH/g-wet cells; 1035 U/g-wet cells) was among the highest productivity reported for recombinant E. coli. With its low K M-value towards glucose (5.5 mM) and NADP(+) (0.05 mM), GluDH-BA was highly suitable for in vivo applications. In this work, a recombinant solvent-tolerant B. subtilis BA overexpressing GluDH-BA was developed and evaluated by coupling with B. subtilis overexpressing an enzyme P450 BM3 F87V for a whole-cell hydroxylation of n-hexane. Significantly higher products obtained clearly proved that B. subtilis BA was an effective cofactor regenerator, a valuable asset for bioproduction of value-added chemicals.

Construction of CoA-dependent 1-butanol synthetic pathway functions under aerobic conditions in Escherichia coli.

1-Butanol is an important industrial platform chemical and an advanced biofuel. While various groups have attempted to construct synthetic pathways for 1-butanol production, efforts to construct a pathway that functions under aerobic conditions have met with limited success. Here, we constructed a CoA-dependent 1-butanol synthetic pathway that functions under aerobic conditions in Escherichia coli, by expanding the previously reported (R)-1,3-butanediol synthetic pathway. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, bld (butylraldehyde dehydrogenase) from Clostridium saccharoperbutylacetonicum, and inherent alcohol dehydrogenase(s) from E. coli. To evaluate the potential of this pathway for 1-butanol production, culture conditions, including volumetric oxygen transfer coefficient (kLa) and pH were optimized in a mini-jar fermenter. Under optimal conditions, 1-butanol was produced at a concentration of up to 8.60gL(-1) after 46h of fed-batch cultivation.

Emulsification efficiency of adsorbed chitosan for bacterial cells accumulation at the oil-water interface.

The use of bacterial cell or biocatalyst for industrial synthetic chemistry is on the way of significant growth since the biocatalyst requires low energy input compared to the chemical synthesis and can be considered as a green technology. However, majority of natural bacterial cell surface is hydrophilic which allows poor access to the hydrophobic substrate or product. In this study, Escherichia coli (E. coli) as a representative of hydrophilic bacterial cells were accumulated at the oil-water interface after association with chitosan at a concentration range of 0.75-750 mg/L. After association with negatively charged E coli having a ζ potential of -19.9 mV, a neutralization of positively charged chitosan occurred as evidenced by an increase in the ζ potential value of the mixtures with increasing chitosan concentration up to +3.5 mV at 750 mg/L chitosan. Both emulsification index and droplet size analysis revealed that chitosan-E. coli system is an excellent emulsion stabilizer to date because the threshold concentration was as low as 7.5 mg/L or 0.00075% w/v. A dramatic increase in the surface hydrophobicity of the E. coli as evidenced by an increase in contact angle from 19 to 88° with increasing chitosan concentration from 0 to 750 mg/L, respectively, resulted in an increase in the stability of oil-in-water emulsions stabilized by chitosan-E. coli system. The emulsion was highly stable even the emulsification was performed under 20% salt condition, or temperature ranged between 20 and 50 °C. Emulsification was failed when the oil volume fraction was higher than 0.5, indicating that no phase inversion occurred. The basic investigation presented in this study is a crucial platform for its application in biocatalyst industry and bioremediation of oil spill.

Enhanced 3-methylcatechol production by Pseudomonas putida TODE1 in a two-phase biotransformation system.

In this study, genetically engineered Pseudomonas putida TODE1 served as a biocatalyst for the bioproduction of valuable 3-methylcatechol (3MC) from toluene in an aqueous-organic two-phase system. The two-phase system was used as an approach to increase the biocatalyst efficiency. Among the organic solvent tested, n-decanol offered several benefits including having the highest partitioning of 3MC, with a high 3MC yield and low cell toxicity. The effect of media supplementation with carbon/energy sources (glucose, glycerol, acetate and succinate), divalent metal cations (Mg(2+), Ca(2+), Mn(2+) and Fe(2+)), and short-chain alcohols (ethanol, n-propanol and n-butanol) as a cofactor regeneration system on the toluene dioxygenase (TDO) activity, cell viability, and overall 3MC yield were evaluated. Along with the two-step cell preparation protocol, supplementation of the medium with 4 mM glycerol as a carbon/energy source, and 0.4 mM Fe(2+) as a cofactor for TDO significantly enhanced the 3MC production level. When in combination with the use of n-decanol and n-butanol as the organic phase, a maximum overall 3MC concentration of 31.8 mM (166 mM in the organic phase) was obtained in a small-scale production, while it was at 160.5 mM (333.2 mM in the organic phase) in a 2-L scale. To our knowledge, this is the highest 3MC yield obtained from a TDO-based system so far.

Optimization of zofimarin production by an endophytic fungus, Xylaria sp. Acra L38.

To optimize the medium for high zofimarin production, sucrose maltose, glucose, tryptone and peptone were used in an orthogonal array design experiment, where the highest value of zofimarin produced was 25.6 µg/mL. This value was about 3 times higher than that obtained with Czapek yeast extract (CzYE) culture medium. A study with Plackett-Burman design showed that sucrose, maltose, glucose and NaNO3 were significant factors in zofimarin production. Further studies using central composite design (CCD) showed the significance of glucose and the interactions of these critical components affecting zofimarin production. Multiple regression analysis of the data yielded a poor fit as shown by the mismatch of the model with these variable factors. When a polynomial equation was applied, the maximum zofimarin production was predicted to be 201.9 µg/mL. Experimental verification yielded a much lower amount of zofimarin, at around 70 µg/mL. Reconsideration of the CCD data and repetition of some runs with high zofimarin production resulted in reproducible zofimarin yield at 79.7 µg/mL. Even though the amount was lower than the predicted value, the medium optimization study was considered to be quite successful as the yield increased to around 8 times that obtained with the original CzYE culture medium.

Optimization of zofimarin production by an endophytic fungus, Xylaria sp. Acra L38

To optimize the medium for high zofimarin production, sucrose maltose, glucose, tryptone and peptone were used in an orthogonal array design experiment, where the highest value of zofimarin produced was 25.6 µg/mL. This value was about 3 times higher than that obtained with Czapek yeast extract (CzYE) culture medium. A study with Plackett-Burman design showed that sucrose, maltose, glucose and NaNO3 were significant factors in zofimarin production. Further studies using central composite design (CCD) showed the significance of glucose and the interactions of these critical components affecting zofimarin production. Multiple regression analysis of the data yielded a poor fit as shown by the mismatch of the model with these variable factors. When a polynomial equation was applied, the maximum zofimarin production was predicted to be 201.9 µg/mL. Experimental verification yielded a much lower amount of zofimarin, at around 70 µg/mL. Reconsideration of the CCD data and repetition of some runs with high zofimarin production resulted in reproducible zofimarin yield at 79.7 µ/mL. Even though the amount was lower than the predicted value, the medium optimization study was considered to be quite successful as the yield increased to around 8 times that obtained with the original CzYE culture medium.