Production of biodegradable polymer by A. eutrophus using volatilefatty acids from acidified wastewater

Wenquan Ruan *, Jian Chen, Shiyi Lun

School of Biotechnology, Wuxi University of Light Industry, Wuxi 214036, China

Received 29 July 2002; received in revised form 9 January 2003; accepted 20 February 2003

Abstract

A new process of production of biodegradable polymer poly(hydroxyalkanoates) (PHAs) was studied with Alcaligenes eutrophus

using volatile fatty acids (VFA) from acidified wastewater. The process was divided into two distinct stages, cell growth with

fructose and PHAs polymerization with VFA. Cell growth was much better with fructose than with VFA. After using fructose for

growth, A. eutrophus utilized VFA as the substrate to polymerization. The concentration of PHAs reached 16.7 g/l by fed-batch

cultivation. Using VFA from acidified wastewater, A. eutrophus had a good performance for polymerization, and the concentration

of cell and PHAs in broth reached 15.9 and 9.6 g/l, respectively.

# 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Poly (hydroxyalkanoates ); Acidification; Fermentation; VFA

1. Introduction

Poly(hydroxyalkanoates) (PHAs), a kind of biode-

gradable polymer, have very attractive properties which

are ideal substitute products of some synthetic plastics.

But PHAs still can not enter into our routine life in the

present time and a major drawback to the industrial

realization of bioplastic PHAs production is its higher

production cost compared with conventional petro-

chemical polymers [1,2]. One of the important factors

in determining the economics of PHAs production on an

industrial scale is the high raw materials price. Among

the substrates required, the carbon source is of prime

significance in the case of PHAs production, since PHAs

are composed only of C, H and O atoms. The main

materials used for the production of PHAs by Alcali-

genes eutrophus are fructose and volatile fatty acids

(VFA) which are both expensive. Significant research

has been focused on using VFA from acidified waste-

water from anaerobic systems [3] by which cheaper raw

materials for the production of PHAs could be obtained.

A. eutrophus first attracted scientific investigation

because of its ability to grow on completely inorganic

nutrients. Doi and colleagues [4] investigated the char-

acteristics of several microorganisms on the production

of PHAs using organic acids as sole carbon source

during fermentation. Lee and Yu [5] verified that A.

eutrophus could use VFA as carbon source to synthesize

PHAs. The VFA used in their experiment were obtained

from an anaerobic system in which biodegradable

components in wastes were digested under anaerobic

conditions by acidogenic bacteria, VFA such as acetic

acid, propionic acid, butyric acid and other soluble

organic compounds could be harvested from the efflu-

ent. Jin and Chen [6] set up a composite anaerobic

acidification�/fermentation system to produce PHAs.

They found butyric acid was the most suitable acid for

A. eutrophus growth and PHAs accumulation. VFA* Corresponding author. Tel./fax: �/86-510-588-8301.

E-mail address: wqruan@public1.wx.js.cn (W. Ruan).

Process Biochemistry 39 (2003) 295�/299

www.elsevier.com/locate/procbio

0032-9592/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0032-9592(03)00074-8

were the carbon source for both growth of microorgan-

ism and polymerization of PHAs in their research.

In this paper, a new process of production of

biodegradable polymer (PHAs) was developed with A.

eutrophus using VFA from acidified wastewater.

2. Materials and methods

2.1. Strain and media

A.eutrophus WSH8 (a mutant strain of A. eutrophus

DSM545) was used in this study. The bacteria werestored on nutrient broth slants with yeast extract 10 g/l,

peptone 10 g/l, meat extract 5 g/l and ammonium sulfate

5 g/l, agar 20 g/l. The strain was activated before using,

and enlarged in flasks containing the medium of

fermentation. In a 2 l fermentor, cells were cultivated

in synthetic medium of the same compositions as in the

literature [5,6] except that fructose, acetic acid, propio-

nic acid, butyric acid were used as a carbon source inthis study.

2.2. Assays

The determination of cell dry weight (DCW) was

made via a dry ice vacuum process. The amount of

PHAs was measured by GC [7]: about 40 mg of dry

bacterial mass was weighed in a tightly sealable vial(volume 10 ml). A 2-ml volume of dichlorine ethane

(DCE), 2 ml propanol containing hydrochloric acid (1

volume concentrated hydrochloric acid�/4 volume pro-

panol) and 200 ul of internal standard solution (2.0 g

benzoic acid in 50 ml propanol) were added and the

whole kept for 3.5�/4 h in an incubator at 80 8C. The

mixture was shaken at the beginning and also during the

incubation from time to time. After cooling to roomtemperature, 4 ml of water is added, and the mixture

shaken for 20�/30 s. The heavier phase (DCE�/propanol)

was injected directly into the gas chromatograph.

Quantitative evaluation was effected by means of the

quotient of the peak areas of hydroxybutyric acid and

benzoic acid.

VFA were measured by GC (Hewlett�/Packard 5890

series II) equipped with a Nukol fused silica capillarycolumn (30 m�/0.25 mm, Supelco, Ballefonte, USA)

and flame ionization detector (FID). Injector and

detector temperatures were at 220 and 250 8C, respec-

tively. The volatile organic compounds were eluted by

helium at 10 ml/min with a temperature program of

10 8C/min from 150 to 190 8C. The water samples were

first filtered through a 0.45 um cellulose nitrate mem-

brane and acidified to pH 3 with concentrated phos-phoric acid prior to GC analysis.

COD concentration was determined using a standard

method. Ammonium sulfate concentration was mea-

sured as the ammonium ion concentration by the

Nesslers reaction [8].

3. Results and discussion

3.1. Characteristic of cell growth of A. eutrophus using

fructose and volatile fatty acid

Jin and Chen [6] found that butyric acid was more

suitable for the polymerization of A. eutrophus thanother VFA, such as acetic acid and propionic acid. For

comparison of the cell growth using both VFA and

fructose, butyric acid was used in the cultivation of A.

eutrophus . Fig. 1 shows the result of cell growth using

fructose and butyric acid. Because of its ill-effects on cell

growth of high concentration of VFA, the concentration

of butyric acid in the start medium was controlled at 10

g/l, and fructose was 20 g/l. Fructose was consumedafter 22 h fermentation and the cell concentration in the

broth reached 12.3 mg/l. With the same cultivation

condition and using butyric acid as the carbon source,

the cell concentration reached only 2.4 mg/l in the broth

after 30 h. Butyric acid also produced a long lag stage of

the cell growth.

The number of cells in broth is an important factor

for the production of PHAs. In order to produce a highconcentration of cells in broth, it is necessary to use

fructose instead of VFA as the carbon source of

cultivation of A. eutrophus . VFA was, therefore, used

for synthesis of PHAs by A. eutrophus .

3.2. PHAs production of A. eutrophus using fructose and

butyric acid

In the fermentation of A. eutrophus , an efficient

system was developed using nitrogen as the limitation

Fig. 1. A. eutrophus growth on fructose and butyric acid. �/j�/,

Fructose; �/m�/, buytric acid; �/I�/, DCW from fructose; �/k�/, DCW

from butyric acid.

W. Ruan et al. / Process Biochemistry 39 (2003) 295�/299296

factor of PHAs production [6,7]. By controlling the

concentration of ammonia in the fermentation broth,

two different stages of A. eutrophus fermentation are

evident, cell growth and PHAs polymerization. With

high concentration of ammonia in broth (balanced

growth conditions), cells mainly use nutrient substrates

to grow and under unbalanced growth conditions

(limitation of ammonia in broth), microorganisms use

most of the nutrient to polymerize PHAs. A fermenta-

tion strategy of A. ertrophus was obtained in this

research by controlling the concentration of ammonia.

In the cell growth stage under conditions of balanced

culture, fructose was used to produce a high concentra-

tion of cells in broth, then the fermentation was

switched to the stage of polymerization by controlling

the ammonia concentration at 70�/90 mg/l (unbalanced

culture). At the beginning of the second stage, VFA

were added into broth as the only carbon source for

polymerization.

Fig. 2 illustrates the time course of variation of DCW,

butyric acid, PHAs and fructose during the fermenta-

tion. The processing was divided into two stages by

controlling the ammonia concentration in broth [6,9,10].

Fructose was digested by A. eutrophus for cell growth

and in the first 20 h, the concentration of fructose in

broth decreased from 20 to 1.8 g/l, and cell concentra-

tion increased to 11.3 g/l. Fructose could not be detected

after 22 h. Butyric acid was added to the broth at 20 h

while the ammonia concentration decreased to below 90

mg/l. The fermentation was switched into the stage of

polymerization. The ammonia ion concentration in

broth was maintained at a level of 60�/80 mg/l during

the polymerization stage. DCW in broth had a small

increment, from 11.3 g/l at 20 h to 17 g/l at 50 h of

fermentation. The concentration of PHAs in broth

increased from 3 to 11.3 g/l. The increasing rate of

PHAs was 0.27 g/l h, much higher than that of cell

growth (0.16 g/l h) during polymerization.

From this experiment, it was clear that this fermenta-

tion process was operative for the production of PHAs.

With a large concentration of cells in broth, a high

concentration of PHAs could be obtained by A.

eutrophus using butyric acid.

3.3. Volatile acids as the production carbon source

The VFA used in this research were acetic acid,

propionic acid, butyric acid. These acids are the main

products of an acidification system of wastewater [3,6].

The polymerizing characteristic of A. eutrophus usingthese three acids was compared in this research.

The experiment was conducted in three individual

flasks after producing sufficient cell concentration from

fermentation with fructose. Fig. 3 illustrates the com-

parison of polymerization of A. eutrophus using three

VFA with an initial concentration 10.5 g/l in broth.

Butyric acid was better for utilization than other two

acids for the polymerization, and propionic acid, theodd carbon acid, better than acetic acid.

3.4. The fermentation process of A. eutrophus by fed-

batch cultivation

The characteristic of growth and polymerization of A.

eutrophus was studied by fed-batch fermentation. Bu-

tyric acid was used as the carbon source of polymeriza-tion by A. eutrophus in this research. Fig. 4 shows the

fermentation processing of A. eutrophus by the fed-

batch cultivation. The acid was added into reactor from

20 to 80 h at 2 ml/h at a concentration of 350 g/l while

ammonium was maintained at a level of 80 mg/l. DCW

increased continuously after 20 h at a rate of 0.222 g/l h

composed with a rate of 0.525 g/l h during the first 20 h.

The cell concentration reached 23 g/l at the end offermentation. The increment of DCW was mainly

attributed to the synthesis of PHAs in cells. The content

of PHAs in cell was about 70% of DCW. The

Fig. 2. The fermentation processing of A. eutrophus with fructose and

butyric acid. �/j�/, Fructose; �/2�/, butyric acid; �/"�/, ammonium;

�/m�/, DCW from fructose; �/'�/, content of PHA.

Fig. 3. Comparsion of polymerization of A. eutrophus using VFA.

�/j�/, Butyric acid; �/"�/, proponic acid; �/'�/, acetic acid.

W. Ruan et al. / Process Biochemistry 39 (2003) 295�/299 297

concentration of PHAs in broth changed from 2.7% at

20 h to 16.65% at the end, with a 5.1-times increase.

3.5. Volatile fatty acids in acidified effluent

Formation of VFA from synthetic glucose wastewater

was studied in a thermophilic (55 8C) upflow anaerobic

sludge blanket (UASB) reactor. The distribution of

organic acids (especially butyric and propionic) in the

effluent was dependent on chemical oxygen demand

(COD) loading rate, pH and hydraulic retention time

(HRT) of wastewater in the reactor. The thermophilicUASB reactor showed a stable performance on hydro-

lysis and acidogenesis of glucose as well as suspended

solid removal at short HRT during operation.

The production of VFA was proportional to COD

loading rate. Fig. 5 shows the performance of acidifica-

tion dependant on HRT in a thermophilic UASB with

artificial wastewater containing 3% glucose. The yield of

VFA based on COD was around 0.28 g VFA/g COD

over the COD loading. Glucose was almost totally

acidified at an HRT of 3 h. The major products in

acidified effluent were acetic acid, lactic acid, butyricacid and propionic acid. The concentration of each acid

varied greatly with change in HRT. Since the best acid

for A. eutrophus cells growth and PHAs accumulation

was butyric acid, the condition of acidification was

controlled for the transfer from glucose to butyric acid.

When the HRT was 3 h, the concentration of lactic acid

was the highest in the effluent, reaching 1.43 g/l,

however, butyric acid was only 0.8 mg/l. As the HRTchanged from 3 to 12 h, lactic acid decreased dramati-

cally, was almost zero at 12 h of HRT, and the

concentration of butyric acid reached 8.6 g/l, higher

than others.

The acidified effluent was centrifuged and then was

concentrated to 150 g/l for use of polymerization.

3.6. Polymerization of A. euthophus with VFA from

acidified effluent

VFA from the thermophilic UASB was concentrated

to 150 g/l which, as feeding substrate, was fed to a 2 l

reactor from 20 to 70 h at the rate of 0.3�/0.5 g/l h. The

residual acids in broth were controlled at the level of 2�/

6 g/l. The fermentation processing with concentrated

acids is shown in Fig. 6. DCW increased from 9.8 g/l at

20 h to 15.9 g/l at the end of fermentation. Accordingly,

the concentration of PHAs in broth changed from 1.9 to9.8 g/l. From the concentration variation of each acid, it

was obvious that butyric acid was the easiest acid for the

polymerization of PHAs by A. eutrophus .

The biosynthesis of PHAs with odd carbon acid or

even carbon acid is different. With even carbon acids

(acetic and butyric acid), A. eutrophus will mainly

synthesis the PHB, and with odd acid (propionic acid)

Fig. 4. The production of PHAs with butyric acid by fed-batch

cultivation. �/"�/, DCW; �/m�/, fructose; �/k�/, residual butyric acid;

�/'�/, PHA; �/^�/, ammonium.

Fig. 5. The distribution of VFA in anaerobic reactor as the change of

HRT. �/2�/, Glucose; �/'�/, butyric acid; �/j�/, acetic acid; �/m�/,

lactic acid; �/"�/, proponic acid.

Fig. 6. The processing of PHAs production by feeding VFA. �/"�/,

DCW; �/m�/, ammonium; �/^�/, proponic acid; �/'�/, PHA; �/k�/,

butyric acid; �/j�/, fructose; �/I�/, acetic acid.

W. Ruan et al. / Process Biochemistry 39 (2003) 295�/299298

the cell will produce both HB and HV sections together

that make biopolymer more flexible and more compe-

titive. Fig. 7 describes the situation of PHAs, HB and

HV which were synthesized by A. eutrophus withconcentrated acidified effluent contained the odd carbon

acid�/propionic acid. The content of HV section in-

creased in polymer during the fermentation. The ratio of

HV:HB was about 1:10.1 at the end of the processing. It

will be quite significant to obtain more propionic acid in

acidified effluent for the better properties of PHAs

products.

4. Conclusion

A. eutrophus had the ability to use fructose to grow

and VFA to polymerize. A new process of cell growth

with fructose and PHAs polymerization with VFA was

efficient. The most suitable organic acid utilized for

PHAs accumulation was butyric acid, the concentrationof PHAs reached 11.2 g/l, higher than propionic acid

and acetic acid. VFA could be obtained from an

acidified thermophilic UASB reactor, which was con-

trolled for the formation of butyric acid in effluent. The

yield of PHAs increased greatly during the fed-batch

fermentation with concentrated VFA from acidified

wastewater by A. eutrophus , the concentrations of

DCW and PHAs were 15.9 and 9.6 g/l, respectively.

Acknowledgements

The authors thank National Natural Science Founda-

tion of China for providing financial support.

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W. Ruan et al. / Process Biochemistry 39 (2003) 295�/299 299