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Biochemical Engineering Journal 78 (2013) 80– 84

Biochemical Engineering Journal 78 (2013) 80– 84 Contents lists available at SciVerse ScienceDirect Biochemical Engineering Journalric acid could o btain 36–51 wt % and Correspo n d i n g a u t h o r . T e l . : + 8 8 6 3 9 3 1 7 4 9 7 ; f a x : + 8 8 6 39357025. E-mail addresses: bychen@niu.edu.tw , boryannchen@yahoo.com.tw (B.-Y. Chen). 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.04.024 46–69 wt% of high PHA contents, respectively [6,7] . The reason why these PHA-generating bacteria could own such promising capabilities of producing PHA and associated monomers was due to induced expression of their DNA sequences for PHA synthe- sis ( e.g ., PhaA , PhaB , PhaC ; [8] ). In fact, the expression of these genes interacted with each other and could be induced at nutrient limiting conditions to synthesize specific monomers and polymers of PHAs. Moreover, several naturally-occurring microorganisms have capabilities for synthesis of PHAs at conditions of physiolog- ical stress ( e.g ., nitrogen, magnesium limiting conditions; [9] ). In particular, several PHA-producing microbes evolved in contami- nated environments could simultaneously degrade pollutants and synthesize PHAs, since the metabolism of PHA synthesis could be expressed in hostile environments. Due to this, this study tended to explore optimal operation strategy of PHA production using indigenous dye-decolorizing bacterium A. hydrophila NIU01 [10] for wastewater treatment and materials recycling and reuses afterwards. Recently, prior study [11] also showed the promising capability of PHA synthesis for A. hydrophila in the presence of decolorized intermediate(s) for wastewater decolorization. How- ever, optimal strategy of operation to cellular capability of PHA synthesis was remained open to be explored for promising feasi- bility to practical applications. Thus, indigenous dye-decolorizing bacterium A. hydrophila NIU01 was selected to explore whether using coconut oil-bearing MR medium could significantly stimulate synthetic capabilities of PHAs (e.g., poly-3-hydroxybutyrate (PHB), poly-3-hydroxyhexanoate (PHHx) or poly(3-hydroxybutyrate-co- 3-hydroxyhexanoate) (P(HB- co -HHx))). Next, operation strategy to " id="pdf-obj-0-6" src="pdf-obj-0-6.jpg">

Contents lists available at SciVerse ScienceDirect

Biochemical Engineering Journal

j o u r n al hom epa ge: www.elsevier.com/locate/bej

Biochemical Engineering Journal 78 (2013) 80– 84 Contents lists available at SciVerse ScienceDirect Biochemical Engineering Journalric acid could o btain 36–51 wt % and Correspo n d i n g a u t h o r . T e l . : + 8 8 6 3 9 3 1 7 4 9 7 ; f a x : + 8 8 6 39357025. E-mail addresses: bychen@niu.edu.tw , boryannchen@yahoo.com.tw (B.-Y. Chen). 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.04.024 46–69 wt% of high PHA contents, respectively [6,7] . The reason why these PHA-generating bacteria could own such promising capabilities of producing PHA and associated monomers was due to induced expression of their DNA sequences for PHA synthe- sis ( e.g ., PhaA , PhaB , PhaC ; [8] ). In fact, the expression of these genes interacted with each other and could be induced at nutrient limiting conditions to synthesize specific monomers and polymers of PHAs. Moreover, several naturally-occurring microorganisms have capabilities for synthesis of PHAs at conditions of physiolog- ical stress ( e.g ., nitrogen, magnesium limiting conditions; [9] ). In particular, several PHA-producing microbes evolved in contami- nated environments could simultaneously degrade pollutants and synthesize PHAs, since the metabolism of PHA synthesis could be expressed in hostile environments. Due to this, this study tended to explore optimal operation strategy of PHA production using indigenous dye-decolorizing bacterium A. hydrophila NIU01 [10] for wastewater treatment and materials recycling and reuses afterwards. Recently, prior study [11] also showed the promising capability of PHA synthesis for A. hydrophila in the presence of decolorized intermediate(s) for wastewater decolorization. How- ever, optimal strategy of operation to cellular capability of PHA synthesis was remained open to be explored for promising feasi- bility to practical applications. Thus, indigenous dye-decolorizing bacterium A. hydrophila NIU01 was selected to explore whether using coconut oil-bearing MR medium could significantly stimulate synthetic capabilities of PHAs (e.g., poly-3-hydroxybutyrate (PHB), poly-3-hydroxyhexanoate (PHHx) or poly(3-hydroxybutyrate-co- 3-hydroxyhexanoate) (P(HB- co -HHx))). Next, operation strategy to " id="pdf-obj-0-17" src="pdf-obj-0-17.jpg">

Exploring two-stage fermentation strategy of polyhydroxyalkanoate production using Aeromonas hydrophila

Biochemical Engineering Journal 78 (2013) 80– 84 Contents lists available at SciVerse ScienceDirect Biochemical Engineering Journalric acid could o btain 36–51 wt % and Correspo n d i n g a u t h o r . T e l . : + 8 8 6 3 9 3 1 7 4 9 7 ; f a x : + 8 8 6 39357025. E-mail addresses: bychen@niu.edu.tw , boryannchen@yahoo.com.tw (B.-Y. Chen). 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.04.024 46–69 wt% of high PHA contents, respectively [6,7] . The reason why these PHA-generating bacteria could own such promising capabilities of producing PHA and associated monomers was due to induced expression of their DNA sequences for PHA synthe- sis ( e.g ., PhaA , PhaB , PhaC ; [8] ). In fact, the expression of these genes interacted with each other and could be induced at nutrient limiting conditions to synthesize specific monomers and polymers of PHAs. Moreover, several naturally-occurring microorganisms have capabilities for synthesis of PHAs at conditions of physiolog- ical stress ( e.g ., nitrogen, magnesium limiting conditions; [9] ). In particular, several PHA-producing microbes evolved in contami- nated environments could simultaneously degrade pollutants and synthesize PHAs, since the metabolism of PHA synthesis could be expressed in hostile environments. Due to this, this study tended to explore optimal operation strategy of PHA production using indigenous dye-decolorizing bacterium A. hydrophila NIU01 [10] for wastewater treatment and materials recycling and reuses afterwards. Recently, prior study [11] also showed the promising capability of PHA synthesis for A. hydrophila in the presence of decolorized intermediate(s) for wastewater decolorization. How- ever, optimal strategy of operation to cellular capability of PHA synthesis was remained open to be explored for promising feasi- bility to practical applications. Thus, indigenous dye-decolorizing bacterium A. hydrophila NIU01 was selected to explore whether using coconut oil-bearing MR medium could significantly stimulate synthetic capabilities of PHAs (e.g., poly-3-hydroxybutyrate (PHB), poly-3-hydroxyhexanoate (PHHx) or poly(3-hydroxybutyrate-co- 3-hydroxyhexanoate) (P(HB- co -HHx))). Next, operation strategy to " id="pdf-obj-0-23" src="pdf-obj-0-23.jpg">

Bor-Yann Chen a, , Jhao-Yin Hung a , Tz-Jau Shiau a , Yu-Hong Wei b

a Department of Chemical and Materials Engineering, National I-Lan University, 1 Shan-Lung Road, I-Lan, 26047, Taiwan b Institute of Biotechnology and Bioengineering, Yuan-Ze University, Chungli, Taoyuan 320, Taiwan

a r

t i

c l e

i n f o

Article history:

Received 26 October 2012 Received in revised form 21 April 2013

Accepted 26 April 2013

Available online 6 May 2013

Keywords:

Bioremediation Biostimulation Fermentation Polyhydroxyalkanoates (PHAs)

Aeromonas hydrophila

Poly-3-hydroxybutyrate (PHB)

a b s t r a c t

With consideration of sustainable development, this study explored the fermentation strategy of cost-effective production of biodegradable polymer- polyhydroxyalanoates (PHAs) for feasibility of eco- friendly materials recycling during wastewater treatment. As prior studies showed that Aeromonas hydrophila NIU01 was a promising PHA-producing bacterium, this follow-up study tended to seek for optimal nutrient-supplementation strategy to stimulate maximal PHA accumulation of A. hydrophila

NIU01 for cellular production. As maximal PHA production took place at growth-limiting conditions, two-stage fermentation was much more appropriate for practical applications compared to batch mode of operation. Moreover, this optimal two-stage operation strategy maximized cellular PHA production under nitrogen-limiting conditions at C/N molar ratio of 60/1. For materials recycling, this operation strategy could be applicable to simultaneous PHB production and wastewater decolorization using A. hydrophila.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

As most of popularly-used plastics are not biodegradable, approx. 25 billions tons plastics significantly accumulated in world- wide natural environment. In fact, “plastic pollution has become a man-made global catastrophe” (e.g., “plastic ocean” in North Pacific and Atlantic Ocean) nowadays [1]. Polyhydroxyalkanoates (PHAs) as one of biomaterials to replace plastics are intracellular energy storage linear polyesters produced in nature by bacte- rial fermentation of sugar and lipids. Due to biocompatibility and biodegradability of biologically-produced PHA for possible uses as plastics with promising physical and chemical characteristics (e.g., can be changed by blending and/or modifying the surface), PHAs could be applicable to be biodegradable polymers for green tech- nology of sustainable development [2]. As a matter of fact, several naturally-occurring microbes were reported to have capabilities of PHA biosynthesis (e.g., Azotobacter vinelandii [3], Pseudomonas sp. [4] and Aeromonas sp. [5]). Regarding the bacteria that gener- ated PHAs in different monomer compositions via diverse synthetic pathways for fermentative production, Aeromonas hydrophila, Pseu- domonas aeruginosa and P. putida were often popularly studied [4]. For example, wild-type and mutant strains of A. hydrophila cultured at 4 or 8 g L 1 lauric acid could obtain 36–51 wt % and

Corresponding author. Tel.: +886 39317497; fax: +886 39357025. E-mail addresses: bychen@niu.edu.tw, boryannchen@yahoo.com.tw (B.-Y. Chen).

1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.bej.2013.04.024

46–69 wt% of high PHA contents, respectively [6,7]. The reason why these PHA-generating bacteria could own such promising capabilities of producing PHA and associated monomers was due to induced expression of their DNA sequences for PHA synthe- sis (e.g., PhaA, PhaB, PhaC; [8]). In fact, the expression of these genes interacted with each other and could be induced at nutrient limiting conditions to synthesize specific monomers and polymers of PHAs. Moreover, several naturally-occurring microorganisms have capabilities for synthesis of PHAs at conditions of physiolog- ical stress (e.g., nitrogen, magnesium limiting conditions; [9]). In particular, several PHA-producing microbes evolved in contami- nated environments could simultaneously degrade pollutants and synthesize PHAs, since the metabolism of PHA synthesis could be expressed in hostile environments. Due to this, this study tended to explore optimal operation strategy of PHA production using indigenous dye-decolorizing bacterium A. hydrophila NIU01 [10] for wastewater treatment and materials recycling and reuses afterwards. Recently, prior study [11] also showed the promising capability of PHA synthesis for A. hydrophila in the presence of decolorized intermediate(s) for wastewater decolorization. How- ever, optimal strategy of operation to cellular capability of PHA synthesis was remained open to be explored for promising feasi- bility to practical applications. Thus, indigenous dye-decolorizing bacterium A. hydrophila NIU01 was selected to explore whether using coconut oil-bearing MR medium could significantly stimulate synthetic capabilities of PHAs (e.g., poly-3-hydroxybutyrate (PHB), poly-3-hydroxyhexanoate (PHHx) or poly(3-hydroxybutyrate-co- 3-hydroxyhexanoate) (P(HB-co-HHx))). Next, operation strategy to

B.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 80– 84

81

optimize performance of such cradle-to-cradle practical operation will be explored. Recently, Wei et al. [12] implemented two-stage fermentation strategy for Cupriavidus taiwanensis to increase PHB production from 9.67 wt% (shake-flask culture) to 72% (two-stage fermentation). As this study tends to explore operation strategy for materials recycling during dye-containing wastewater treatment, dye-decolorizer Aeromonas hydroiphila NIU01 which also owned significant tolerant capabilities to dyes and decolorized interme- diates was used for study, but not popularly-used PHB-producing bacteria (e.g., Ralstonia eutropha) [11]. This feasibility study showed that separation cell growth phase from PHA production phase of A. hydrophila NIU01 using two-stage fermentation strategy to maxi- mize PHA production was technically viable.

  • 2. Materials and methods

    • 2.1. Microbial cultures

This feasibility study used indigenous pollutant-degrading bacteria isolated from Cross-Strait Taiwan and Mainland China [13]. All strains were cultured in LB broth medium for 12–16 h and then the cultures were mixed with 40% (v/v) glycerol to store at 80 C for long-term use.

  • 2.2. Culture media

PHA production (data not shown). After batch fermentation, cul- tured broths were aseptically cooled to 4 C in order to completely solidify lauric acid (C12). Next, filter papers (ADVANTEC, NO. 5A, 7 mm) were used to separate biomass from solid particles of lauric acid. To remove residual abiotic ash from biomass, biomass solu- tions were three-times washed with distilled and deionized water, centrifuged (10,000 rpm, 10 min), and then the supernatants were discarded. The harvested biomass was then dried at 100 C until constant weight was achieved for PHA-content analysis afterwards.

  • 2.5. Two-stage fermentation for PHB production

To avoid additional production loading during microbial growth in the early stage of fermentation, the fermentation (Fermenter FIRSTEK, FB-6S) started on batch mode of operation. Once cell growth reached late exponential growth phase, second stage (production stage) of two-stage fermentation was switched on for the feasibility study (i.e., pH 7.0, 30 C, 200 rpm, 1 vvm (3 LPM) for 3 L working volume fermentation). The model of two-stage fermentation operation was to inspect whether separation of cell growth phase from production phase is technically viable to improve the performance of PHB synthesis. The nutrient substrate – coconut oil (ca. 49.47% lauric acid) was provided in 2–3 h, as this was sufficient to support desired cellular growth and PHB production for study [16].

The basic culture media- MR medium [10], nitrogen-limited

MR medium [14], phosphorus-limited MR medium [5] (per liter) contained 4.0 g (NH 4 ) 2 HPO 4 , 0.8 g MgSO 4 ·7H 2 O, 0.8 g citric acid and 6.67 g KH 2 PO 4 , 10 ml TES-I and 1 ml TES-II. Trace-element solution I (TES-I) (in 1.0 L) contained 5.0 g Fe(III)–NH 4 –citrate and 2.0 g CaCl 2 ·2H 2 O. Trace-element solution II (TES-II) (prepared in 1.0 N HCl) contained 100 mg ZnSO 4 ·7H 2 O, 30 mg MnCl 2 ·4H 2 O,

300

mg

H 3 BO 3 , 200 mg

CoCl 2 ·6H 2 O, 10 mg CuSO 4 ·5H 2 O, 20 mg

NiCl 2 ·6H 2 O and 30 mg Na 2 MoO 4 ·2H 2 O. For two-stage fermenta-

tion, MR medium was used for cell growth in first-stage culture. Nitrogen-limited or phosphorus-limited MR media were adopted for PHA production in second stage culture.

  • 2.3. Analysis of PHA/PHB content

Cultured biomass of 0.020 g dry cell weight was completely mixed with 2.0 mL methanol (in 1% (w/w) sulfuric acid) and 2.0 mL chloroform, and then the mixture was stored in well-sealed test tubes. Test-tube mixtures were incubated in 100 C oven for 6 h reaction to dehydrate intracellular poly-3-hydroxybutyrate (PHB) as methyl(R)-3-hydroxybutyrate (MHB) monomers. After complete reaction, the mixture was cooled down to room temperature. Then, 1 mL 1 N sodium chloride was added to salt-out residual MHB still contained in aqueous phase. For calibration of measurement of PHB content, 350 L internal standard (1% (w/w) benzoic acid dis- solved in chloroform) was added. After static incubation, samples in organic phase of the mixed solutions were chosen for quanti- tative assessment of PHB. Detailed determination of PHA contents was described in Lo et al. [15].

  • 2.4. Batch fermentation

A loopful of pure seed culture taken from single colony on a LB streak plate was inoculated into 50 ml sterilized Luria–Bertani (LB) broth medium for 12–16 h overnight (O/N) preculture. Biomass inoculated into 100 ml nitrogen-limited or phosphorus-limited MR medium containing TES-I and TES-II and lauric acid for 72 h cul- ture at 30 C, 200 rpm. Note that compared to other carbon sources lauric acid was found to be more appropriate carbon source for

  • 3. Results and discussion

    • 3.1. Optimal C/N ratio for cell growth

To have maximal growth capability and minimal production burden for achieving optimal growth rates of economically-feasible bacterial cultures, optimal C/N ratio of nutrient sources should be inspected. As a matter of fact, when lauric acid was used as the nutrient source, it can be metabolized to (R)-3-hydroxyacyl-CoA (3HB-CoA) which is utilized as energy substrate for PHA syn- thesis in the last step of PHA synthesis [17,18]. When C/N ratio decreased (i.e., nitrogen substrate- ammonium sulfate was suffi- ciently provided herein), the mole flux of acetyl-CoA reaching TCA cycle increased and mole fraction of 3HB units relatively decreased. However, at higher C/N ratios such high carbon-substrate concen- trations inhibited capabilities of microbial growth and apparently repressed PHB synthesis. In addition, relatively lowering nitro- gen concentration could promote capabilities of PHA-synthesizing bacteria for the conversion to PHB [19]. Moreover, Grothe et al. [20] also mentioned that the sensitivity of cell and PHB yields to changes in C/N ratio depends upon the type of the nitrogen source used. These explained why optimal C/N ratio was crucial for maximal PHB production as shown herein. When nitrogen-source level in basic MR medium was fixed, comparative cultures for cell growth and PHB production of NIU01 at various C/N ratios were conducted to disclose optimal conditions. As literature mentioned [10], carbon substrate provided in excess could stimulate intra- cellular accumulation of PHAs due to limiting conditions of other nutrient sources. Thus, Fig. 1 indicated that at C/N ratio of 9.89/1 (i.e., 10 g L 1 lauric acid) optimal dry cell weight, PHB content and PHB concentration could be obtained at 4.05 g L 1 , 36.02 wt% and 1.46 g L 1 , respectively. Similar optimal C/N ratio of 10/1 for PHB production was also found for oleic acid; however, cellular produc- tion performance was relatively lower (ca. 10.33% PHB content, dry cell weight 0.99 g L 1 and PHB concentration 0.11 g L 1 ). Recently, Wei et al. [12] showed that at C/N ratio 8/1 maximal PHB produc- tion could be achieved for C. taiwanensis 184 (i.e., 4.15 g L 1 dry cell weight, PHB content 58.81 wt% and PHB concentration 2.44 g L 1 ). Regarding A. hydrophila, when excess carbon source was provided

  • 82 B.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 80– 84

5 40 1.8 (A) 1.6 4 1.4 30 1.2 3 1.0 20 2 0.8 0.6 1
5
40
1.8
(A)
1.6
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Dry cell weight (g L -1 )
Dry cell weight (g L -1 )
PHB content (wt%)
PHB content (wt%)
Fig. 2. Comparison of performances of cellular growth and PHB production of Aeromonas hydrophila NIU01 using
Fig. 2. Comparison of performances of cellular growth and PHB production of
Aeromonas hydrophila NIU01 using 10 g L −1 glycerol, butyric acid (C4), sodium glu-
conate (C6), hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10), lauric acid
PHB concentration (g L -1 )
PHB concentration (g L -1 )

(C12) and oleic acid (C18) as sole carbon source, where samples were taken at 72 h.

C/N rat io (mol/mol)

Fig. 1. Comparative profiles of cell growth and PHA production of Aeromonas hydrophila NIU01 in MR medium at various C/N ratios using (A) lauric acid and (B) oleic acid as sole carbon source (sampled at 72 h).

(e.g., C/N ratio at 20/1), reduction of cell growth and PHB pro- duction was found very likely due to substrate inhibition (i.e., dry cell weight 1.69 g L 1 , PHB content 52.77 wt% and PHB concentra- tion 0.89 g L 1 ). To prevent such inhibitory responses to microbial growth and PHA production, 9.89/1 (ca. 10/1) was thus selected herein for C/N ratio of first-stage in two-stage fermentation.

  • 3.2. Effect of carbon sources

As Chen [21] mentioned, acetyl-CoA is the key component to supply the 3-hydroxyalkanoyl-CoA of various lengths as sub- strates for PHA synthases of diverse specificities. In fact, fatty acids of different chain lengths could provide such varieties of 3-hydroxyalkanoyl-CoA via -oxidation of pathway II. After fatty acid -oxidation, acyl-CoA enters PHA monomer synthesis process. That is, fatty acids of various chain lengths might also be feasi- ble substrates for PHA production. Although lauric acid seemed to be a promising substrate for PHB synthesis [10,11], it is still not a cost-effective carbon source of biostimulation for industrial applications. As coconut oil is rich in lauric acid (ca. 49.47% (w/w)), coconut oil is thus considered herein as alternative carbon source for economically-feasible PHB synthesis [22]. Moreover, coconut oil also contained fatty acids of carbon number 6, 8, 10, 12 (C6, C8, C10, C12 for short) for possible PHB-synthesizing sources. As shown in Fig. 2, apparently fatty acids of higher carbon numbers (i.e., only lauric acid and oleic acid) were more appropriate to be such energy substrates (i.e., C12) for PHB synthesis. It was also sus- pected that lauric acid was optimal carbon-number nutrient source for PHB production of NIU01. Thus, when coconut oil was used as substrates for PHB production, the major carbon source for PHB synthesis can be assumed to be lauric acid and less likely carbon sources are higher carbon-numbered fatty acids (e.g., oleic acid).

  • 3.3. Batch fermentation

To consider possible industrial applications of PHA produc- tion using more cost-effective sources, batch fermentation was

first carried out. Dierick et al. [22] mentioned that coconut oil which was rich in saturated and long-chain fatty acid contained ca. 49.47 g lauric acid/100 g coconut oil. Thus, this study chose coconut oil (ca. 20.21 g L 1 10 g L 1 lauric acid) as an economi- cally feasible carbon source for bacterial fermentation to compare performance of PHB production. The finding (Fig. 3) indicated that using coconut oil as carbon and energy source of A. hydrophila NIU01 could express better efficiency of PHB production (ca. dry cell weight 7.31 g L 1 , PHB content 49.63 wt% and PHB concentra- tion 3.63 g L 1 ); apparently, this was better than prior result (i.e., dry cell weight 4.05 g L 1 , PHB content 36.02% and PHB concen- tration 1.46 g L 1 ). Since coconut oil contained significant amounts of other long-chain fatty acids as possible energy and nutrient sources, this was possibly why a better microbial growth and PHB production could be achieved. Recently, Wei et al. [11] mentioned that operation strategy of continuous fermentation could enhance microbial capability of PHB production. Thus, two-stage fermen- tation was considered as a mode of operation (i.e., first-stage for microbial growth and second for PHB production) for cost-effective of industrial PHA production. To prevent a “metabolic burden” of PHA production at cell growth phase, separation of cell growth from PHA production should be accomplished by proper manipulation of environmental condition(s) (e.g., nitrogen-limiting cultures) to maximize PHA production at cell production phase.

20 80 20 15 60 15 10 40 10 5 20 5 0 0 0 0
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0
0
0
0
100
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500
Dry cell weight (g L -1 )
PHB content (wt%)
PHB concentration (g L -1 )

Time (h)

Fig. 3. Time series profiles of cell growth and PHA production of batch fermentation of Aeromonas hydrophila NIU01 in coconut oil-bearing MR medium at various C/N ratio of 10/1.

B.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 80– 84

83

80 80 2.5 12 2.0 60 10 60 8 1.5 40 40 6 1.0 4 20
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60
80
100
C/N ratio (m ol/mol)
PHHx content (wt%)
Dry cell weight (g L -1 )
PHB content (wt%)
PHAs concentration (g L -1 )

Fig. 4. Comparative profiles of cell growth and PHA production of A. hydrophila NIU01 in nitrogen-limited MR medium at various C/N ratios (measured at 72 h).

  • 3.4. Optimal C/N ratio for PHA production

As Wei et al. [12] mentioned that using two-stage fermen-

tation strategy C. taiwanensis could increase production yield

from 9.67 wt% (shake-flask culture) to ca. 72%. Thus, prior to

implementation of two-stage fermentation in this study, essential

nutrient-limiting (e.g., nitrogen, phosphorus-limiting) condition

should be determined for optimal condition of second-stage PHA

production. Once 12 h shake flask culture reached at late exponen-

tial growth phase in LB medium, cell broth was centrifuged and

supernatant was discarded to reserve cells for initiation of second

stage cell culture. Then, nitrogen-limited or phosphorus-limited

MR media with lauric acid at various levels were supplemented

to inspect optimal C/N and C/P ratio for PHA production. As shown

in Fig. 4, the optimal C/N ratio was obtained at 60/1 using nitrogen-

limiting MR medium for maximal PHA production (4.03 g L 1

dry cell weight, PHB content 53.59 wt% and PHB concentration

2.27 g L 1 ). However, treatability studies to determine optimal C/P

rations showed that phosphorous limiting condition (i.e., C/P ratio

at 30/1) seemed not to be viable to maximize PHA production

(i.e., dry cell weight 1.71 g L 1 , PHB content 8.26 wt% and PHB

concentration 0.41 g L 1 ; data not shown). These indicated that

nitrogen-limited MR medium in C/N ratio of 60/1 was optimal for

PHB production in second stage culture.

  • 3.5. Two-stage fermentation

Due to different metabolic pathways dealing with cell growth

(e.g., nitrogen-sufficient conditions) and PHB synthesis (e.g.,

nitrogen-limited cultures), maximal growth rate should first be

maintained in exponential growth phase to effectively achieve

more appropriate cell population for PHB production. Next, optimal

nutrient-limited condition(s) for PHB synthesis could be applied

at early stationary growth phase for target-product formation

[12,23]. Thus, to separate cell growth from PHB production of

bacterial fermentation, MR medium was used for culture at first-

stage using 20.1 g L 1 coconut oil (i.e., C:N = 10:1) and tryptone

5.0 g L 1 as augmented energy sources to stimulate performance

of cell growth. Then, once cells reached early stationary growth

phase, culture broth was replaced via cell-filtration and nitrogen-

limited MR medium supplemented with coconut oil at various

C/N ratios was added to stimulate promising PHB production.

With such a separation strategy of growth and production phases

for C/N ratio of 60/1, promising cell growth and PHB production

could be achieved (i.e., dry cell weight 16.8 g L 1 , PHB content

62.1 wt%, PHB concentration 10.4 g L 1 at ca. 300 h of second-stage

culture; Fig. 5). The average PHB concentration can be determined

to be

t 0

t f

[PHB]dt

=

t f t 0 t 0

1

t f

t f

t 0

dt

[PHB]dt

=6.34 g L 1 . In contrast, for C/N

ratios of 40/1 and 90/1 at 300 h of second-stage culture, dry cell

20 20 80 C/N 10 C/N 40 15 60 15 10 40 10 5 20 5
20
20
80
C/N 10
C/N 40
15
60
15
10
40
10
5
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C/N 60
60
15
15
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C/N 90
15
60
15
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40
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20
5
0
0
0
0
100
200
300
400
Dry cell weight (g L -1 )
Dry cell weight (g L -1 )
Dry cell weight (g L -1 )
PHB content (wt%)
PHB content (wt%)
PHB content (wt%)
PHB concentration (g L -1 )
PHB concentration (g L -1 )
PHB concentration (g L -1 )

Time (h)

Fig. 5. Comparison on time-series profiles of microbial growth and PHB production for two-stage fermentation of Aeromonas hydrophila NIU01. Dashed lines denoted the times to start second-stage fermentation (i.e., removal of MR medium to sup- plement nitrogen-limited MR medium at C/N molar ratio of 40/1, 60/1 and 90/1).

weight, PHB content, and concentrations were 9.00 and 11.03 g L 1 ,

51.33 and 54.45%, and 4.62 and 5.67 g L 1 , respectively. The cor-

responding average PHB concentrations were 3.90 and 3.80 g L 1 ,

respectively. Thus, the optimal C/N ratio of 60/1 for PHB synthesis

(Fig. 5) was also confirmed with batch data using nitrogen-limited

medium as aforementioned in Fig. 4. This study suggested that two-

stage fermentation was a technically-feasible mode of operation

to stimulate significant accumulation of target product(s) when

cell growth was reached in stationary phase. Follow-up studies

will explore the feasibility of using trace metals for supplemen-

tation and fed-batch culture with exponentially-feeding strategy

to reach optimal of high cell-density cultivation for maximal

PHB production [20]. In addition, as strain NIU01 is a promis-

ing dye-decolorizing and bioelectricity-generating bacterium, the

strategy of PHB production and power generation for materials and

energy recycling during dye-bearing wastewater treatment will be

conducted afterwards for considerations of cradle-to-cradle envi-

ronmental bioremediation.

4. Conclusion

This feasibility study revealed that two-stage fermentation to

effectively separation of cell growth from PHB production to max-

imize PHB concentration was promising. Follow-up studies would

consider simultaneous wastewater treatment and PHB production

for practical applications in materials recycling and reuses.

  • 84 B.-Y. Chen et al. / Biochemical Engineering Journal 78 (2013) 80– 84

Acknowledgements

Financial supports (NSC 98-2221-E-197-007-MY3, NSC 100-

2621-M-197-001, NSC 101-2221-E-197-020) from Taiwan’s

National Science Council and Industrial Technology Research

Institute (ITRI B200-101-YG-02) for this research conducted in

Biochemical Engineering Laboratory sdg , Department of Chemical

much appreciated.

References

255–261.

144–150.

[9] K. Hong, G. Chen, P. Fu Yu, G. Zhang, Y. Liu, H. Chua, Effect of C:N molar ratio on monomer composition of polyhdroxyalk anoates produced by Pseudomonas mendocina 0806 and Pseudomonas pseudoalkaligenus YS1, Appl. Biochem. Biotechnol. 84–86 (2000) 971–980. [10] B.-Y. Chen, T.-J. Shiau, Y.-H. Wei, W.-M. Chen, B.-H. Yu, C.-Y. Yen, C.-C. Hsueh, Feasibility study of polyhydroxyalkanote production for materials recycling using naturally occurring pollutant degraders, J. Taiwan Inst. Chem. Eng. 43 (2012) 455–458.

[11]

B.-Y. Chen, T.-J. Shiau, Y.-H. Wei, W.-M. Chen, Feasibility study on polyhydroxy-

1205–1215.

1381–1386.

[22]

Monographs, Vol. 14, Springer-Verlag, Berlin, 2010, pp. 17–37. N.A. Dierick, J.A. Decuypere, K. Molly, E. Van Beek, E. Vanderbeke, The combined use of triacylglycerols (TAGs) containing medium chain fatty acids (MCFAs) and exogenous lipolytic enzymes as an alternative to nutritional antibiotics in piglet nutrition: II. In vivo release of MCFAs in gastric cannulated and slaughtered piglets by endogenous and exogenous lipases effects on the luminal gut flora and growth performance, Livestock Prod. Sci. 76 (2002) 1–16.

[23] F. Wang, S.Y. Lee, Poly(3-hydroxybutyrate) production with high produc- tivity and high polymer content by a fed-batch culture of Alcaligenes latus under nitrogen limitation, Appl. Environ. Microbiol. 63 (1997)

3703–3706.