• Microbiology and Biotechnology Letters
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• v.26 no.3
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• pp.257-263
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• 1998

Alcaligenes eutrophus was successfully recovered from high cell density broth by pre-treatment with Fe-based coagulants. An inorganic coagulant, Fe$_2$(SO$_4$)$_3$, and a polymerized coagulant, Ferix-3, were used. Good coagulation was observed in broad pH range of 3 to 13, the floe size was increased with increasing pH of culture broth. The optimum pH of fermentation broth for cell recovery was 10 to 13. The optimum coagulant dosages to recover cells with 95% cell recovery were increased with increasing cell concentration. Optimal coagulant dosage was lower when the polymerized coagulant was used rather than the inorganic coagulant. The coexistence of NH$_4$$\^$+/ was increased coagulant requirement, and the coagulant requirement was 0.066g Fe$_3$$\^$+//g NH$_4$$\^$+/.

• Journal of Microbiology and Biotechnology
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• v.3 no.2
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• pp.115-122
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• 1993

Poly(3-hydroxybutyrate)(P(3HB)) accumulation under nutrient-rich condition with various amounts of $(NH_4)_2 SO_4$ was systematically investigated. The results of the electron-microscopy and the solvent extraction showed that the P(3HB) accumulation is unavoidable even under nutrient-rich condition. This indicates that in a two-step culture of Alcaligenes eutrophus H16, the researches should be careful in interpreting the data of polyhydroxyalkanoates(PHAs) accumulation in terms of the carbon-source fed in the second step because the two-step culture product contains the P(3HB) produced under nutrient-rich condition. The polyester production capability in a two-step batch culture of A. eutrophus H16(ATCC 17699) was also investigated using various saccharides and their derivatives such as glucose, fructose, gluconic acid, glucaric acid, sorbitol, lactose, galactose, and mannose. The polyesters synthesized were characterized by 500 MHz$^{1}H-NMR$ spectroscopy, intrinsic viscosity$[\eta]$ measurement in chloroform and differential scanning calorimetry(DSC). 500 MHz $^{1}H-NMR$ analysis showed that all polyesters synthesized generally contained 1~2 mol% of 3HV. Another finding is that the glucose utilization can be increased by changing the autoclaving procedure of the substrate to enhance the P(3HB) production yield up to 46 wt% of P(3HB) in dry cells.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.215-222
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• 1997

The regulatory mechanism of the flux of acetyl-CoA in Alcaligenes eutrophus in unbalanced growth conditions was investigated using a PHB-negative mutant and transformants reintroduced PHB-biosynthesis enzymes through the transformation of cloned phbCAB genes. The PHB-negative mutant was defected absolutly in PHB synthase but partially in ${\beta}$-ketothiolase and acetoacetyl-CoA reductase, and excreted substantial amount of pyruvate to culture broth at late growth phase. The excretion was due to the inhibitory effect of acetyl-CoA on the activity of pyruvate dehydrogenase. The cloned phbC and phbCAB genes were transformed to the PHB-negative mutant strain to reintroduce PHB biosythesis enzymes. Pyruvate excretion could be decreased substantially but not completely by transformation of PHB synthase alone, while pyruvate excretion was ceased by transformation of all three PHB biosynthesis enzymes. To identify the most critical PHB biosynthesis enzyme influencing on the flux of acetyl-CoA, the effect of the variation of PHB biosynthesis enzymes on pyruvate dehydrogenase was investigated. ${\beta}$-Ketothiolase influenced the activity of pyruvate dehydrogenase more sensitively than PHB synthase. ${\beta}$-Ketothiolase, the first step enzyme of PHB biosynthesis that condense acetyl-CoA to acetoacetyl-CoA, seems to be the major enzyme determining the flux of acetyl-CoA to PHB biosynthesis or TCA cycle, and the rate of PHB biosynthesis in A. eutrophus.

• Microbiology and Biotechnology Letters
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• v.26 no.3
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• pp.206-212
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• 1998

The effects of the pretreatment with coagulants on the recovery efficiency of poly (3-hydroxybutyrate, PHB) synthesized in Alcaligenes eutrophus were investigated. Al-base or Fe-base coagulants, and the dispersion method of 30% hypochlorite solution and chloroform were used as coagulants and PHB recovery method, respectively The recovery efficiency of PHB from the cells harvested with Al-base coagulants at the range from 0 to 1000 mg-Al/L was similar to that from cells harvested without the coagulants. At these conditions, the concentrations of residual aluminium in the purified PHB were below 250 mg-Al/kg-PHB, indicating the effect of residual aluminum on the characteristics of the purified PHB can be insignificant. When the dosage of coagulants was over 1000 mg-Al/L, the PHB recovery remarkably decreased with increasing the coagulant dosage. However, the PHB recovery could be enhanced by the use of 50% hypochlorite solution instead of 30% hypochlorite solution. Even though the reduction of PHB recovery efficiency was not found by using Fe-base coagulants, the purified PHB was stained pale red due to residual iron, These results suggest that the use of Al-base coagulants did not exert bad influence on neither PHB recovery efficiency and PHB purity.

• Microbiology and Biotechnology Letters
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• v.20 no.6
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• pp.668-676
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• 1992

For the purpose of optimizing poly-l3-hydroxybutyrate (PHB) production from Alcaligenes eutrophus, two-stage fed-batch culture was adopted. In this system, specifk growth rate was maximized during the first stage whereas specific production rate was maximized during the second stage. The optimal concentrations of glucose and ammonium chloride were 16.6 and 0.54 g/I in the growth stage and 20.0 and 0.07 g/l in the production stage, respectively. Proportional feedback control considering time lag was suggested for PHB production process and a simulator was developed for real-time control purpose.

• Microbiology and Biotechnology Letters
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• v.23 no.5
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• pp.588-592
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• 1995

Fed-batch fermentation was used to produce the high concentrations of poly-$\beta $-hydroxybutyrate (PHB) and poly-$\beta $-(hydroxybutyrate-co-hydroxyvalerate) (PHB/V). Specific growth rate ($\mu $), yield of cell from glucose (Y$_{x/s}$) were calculated from the two samples in 3 to 5 hours of interval and they were reflected on the determination of glucose feeding rate to maintain the glucose concentration at around 10 g/l in the culture broth. PHB was accumulated after the nitrogen became limited at 60 g/l of dry cell weight by changing ammonia water to 4N-NaOH solution. As results, the final dry cell weight (DCW) of 170 g/l, PHB of 115 g/l were obtained in 50 hours and the overall productivity was 2.4 g/l$\cdot $h. After PHB accumulation, cosubstrate of glucose and propionic acid (PA) was fed to accumulate PHB/V. But, PA feeding rate was decreased from 3 g/l$\cdot $h to 1 g/l$\cdot $h to prevent PA from accumulating to high level in the broth, which is very inhibitory to the cells. As results, DCW, PHB and PHV were 147.5 g/l, 90 g/l and 8 mole % of hydroxyvalerate, respectively.

• Proceedings of the Zoological Society Korea Conference
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• 1996.10a
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• pp.196.2-196
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• 1996

No Abstract, See Full Text

• Proceedings of the Microbiological Society of Korea Conference
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• 1998.04a
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• pp.47.2-47.2
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• 1998

• KSBB Journal
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• v.13 no.2
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• pp.187-194
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• 1998

For mass production of polyhydroxyalkanoates (PHA), high cell density cultures of Alcaligenes eutrophus by fed-batch culture under phosphate-limitation condition has been investigated. PHA accumulation by the regulation by the regulation of initial phosphate concentration could be automatically induced, and high density cell culture above 200 g/L also could be successfully produced. The production of Poly-$\beta$-hydroxybutyrate (PHB) and dry cell weight increased with increasing the initial phosphate concentration. When the initial concentrations of phosphate were in the ranges of 1.5~4.5 g-PO$_4$/L, PHB and dry cell weight obtained were 83~266 g/L and 61~216 g/L, respectively, and PHB productivity was in the ranges of 1.35~3.10 g/L.h. When a mixture of glucose and propionic acid is used as carbon sources, poly(3-hydroxybutyrate-co-poly-3-hydroxyvalerate), P(3HB-co-3HV), could be also successfully produced under phosphate limitation condition. When the mole ratio of propionic acid to glucose in the feeding solution is 0.22, a final dry cell weight of 150 g/L and a P(3-HB-co-3HV) of 90 g/L were produced. Morphological changes and size distribution of PHB granules synthesized in A. eutrophus under phosphate-limitation condition are determined by TEM during the course of fed-batch. Mean granule diameters of PHB produced are in the range of 0.36~0.39 $\mu$m, and mean cell size was elongated from 0.54~0.59 $\mu$m$\times$ 1.3~1.5 $\mu$m to 0.83~0.89 $\mu$m $\times$2.0~2.3 $\mu$m. Phosphate concentration in media did not affect size distribution of PHB granule and cell.

• Microbiology and Biotechnology Letters
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• v.26 no.6
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• pp.537-544
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• 1998

The cultivation conditions of transformant Alcaligenes eutrophus AER5 harboring cloned phbC gene for mass production of poly (3-hydroxybutyrate-3-hydroxyvalerate)[P(3HB-3HV)] containing high molar fraction of 3-hydroxyvalerate (3-HV) were investigated. In two-stage batch cultivation, transformant accumulated P(3HB-3HV) containing 52.2 mol% of 3HV compared to 30 mol% of parent strain A. eutrophus H16. The increased 3-HV molar fraction was due to the amplified activity of PHB synthase participating in condensation of 3-HB and 3-HV. To increase efficiency of P(3HB-3HV) accumulation, fructose was added along with precursor compound valerate, and total cell mass and P(3HB-3HV) concentrations remarkably increased, but not 3-HV molar fraction. The effect of magnesium ion showed that P(3HB-3HV) concentration and 3-HV molar fraction were significantly increased upto 6.1 g/L and 71.3 mol% at 0.01 g/L of MgSO$_4$, respectively. The efficiency of several pH adjuster, NaOH, NaOH and (NH$_4$)$_2$SO$_4$, and NH$_4$OH, on total cell mass, p(3HB-3HV) concentration, and 3-HV molar fraction was also compared. To overcome the disadvantage of two-stage cultivation, one-stage intermittent fed-batch cultivation was attempted, such a way 10.0 g/L of fructose was supplied for cell growth at initial 36 hr and then 10.0 g/L of valerate and 5.0 g/L of fructose were applied to induce the accumulation of P(3HB-3HV), consequently, 10.4 g/L of P(3HB-3HV) with 38 mol% of 3-HV fraction could be obtained after 72 hr. These results can be used for elucidating cultivation strategy for mass production of P(3HB-3HV) containing high 3-HV molar fraction using transformant A. eutrophus AER5 harboring cloned phbC gene.