Production of PHA by Mixed Cultures - Trends and Biotech Importance

www.elsevier.com/locate/biotechadv

Biotechnology Advances 22 (2004) 261–279

Research review paper

Production of polyhydroxyalkanoates by

mixed culture: recent trends and

biotechnological importance

H. Salehizadeh a,b,*, M.C.M. Van Loosdrecht a

aKluyver Laboratory for Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft,

The NetherlandsbBiotechnology Group, Chemical Engineering Department, Tarbiat Modarres University, Tehran, Iran

Accepted 16 September 2003

Abstract

Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as

carbon/energy or reducing-power storage material in various microorganisms. PHAs have been

attracting considerable attention as biodegradable substitutes for conventional polymers. To reduce

their production cost, a great deal of effort has been devoted to developing better bacterial strains and

more efficient fermentation/recovery processes. The use of mixed cultures and cheap substrates can

reduce the production cost of PHA. Accumulation of PHA by mixed cultures occurs under transient

conditions mainly caused by intermittent feeding and variation in the electron donor/acceptor

presence. The maximum capacity for PHA storage and the PHA production rate are dependent on the

substrate and the operating conditions used. This work reviews the development of PHA research.

Aspects discussed include metabolism and various mechanisms for PHA production by mixed

cultures; kinetics of PHA accumulation and conversion; effects of carbon source and temperature on

PHA production using mixed cultures; PHA production process design; and characteristics of PHA

produced by mixed cultures.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Bioplastic; Mixed culture; Polyhydroxyalkanoates (PHA); Wastewater

0734-9750/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.biotechadv.2003.09.003

* Corresponding author. Chemical Engineering Department, Tarbiat Modarres University, Tehran, Iran. Fax:

+98-21-8006544.

E-mail address: [email protected] (H. Salehizadeh).

H. Salehizadeh, M.C.M. Van Loosdrecht / Biotechnology Advances 22 (2004) 261–279262

1. Introduction

Over the past years, the use of plastics in packaging and other products has exacerbated

the problem of disposal of solid waste. The nondegradable petrochemical plastics

accumulate in the environment at a rate of 25 million tonnes/year (Lee et al., 1991). In

response to the problem and harmful effects of the plastic wastes on the environment, there

is considerable interest in the development of biodegradable plastics (Leaversuch, 1987).

Among the various biodegradable polymer materials, polyhydroxyalkanoates (PHAs)

provide a good fully degradable alternative to petrochemical plastics (Doi, 1990; Anderson

and Dawes 1990). The properties of PHAs are very similar to those of polyethylene (PE)

and polypropylene (PP) (Howells, 1982; Holmes, 1988; Lee, 1996a). Copolymers of the

PHAs poly(hydroxybutyric acid) (PHB) and poly(hydroxyvaleric acid) (PHV) are far less

permeable to oxygen than are polyethylene and polypropylene. This makes PHA

copolymers a better material for food packaging because there is a reduced need for

antioxidant addition.

PHA is a polyester of hydroxyalkanoates that accumulates as carbon/energy or

reducing-power storage material in microbial cells. PHAs are synthesized and accumulated

as intracellular granules usually when there is an essential growth-limiting component

such as nitrogen, phosphate, sulfur, oxygen or magnesium in the presence of excess carbon

source (Lee, 1996b; Anderson and Dawes, 1990; Poirier et al., 1995). Depending on the

substrate provided, many microorganisms can include a wide variety of 3-hydroxy fatty

acids in the PHA. Since the first finding of PHB in 1926 (Lemoigne, 1926), more than 100

different monomer units have been identified as constituents of PHA in >300 different

microorganisms (Lee, 1996b).

PHA has been industrially produced by pure cultures including Alcaligenes latus,

Azotobacter vinelandii, Pseudomonas oleovorans, recombinant Alcaligenes eutrophus and

recombinant Escherichia coli (Lee and Choi, 1998; Grothe et al., 1999; Grothe and Chisti,

2000). With current advances in PHA research, a PHA concentration of more than 80

g�l� 1 and productivity of more than 2 g�l� 1 h� 1 have been obtained in the laboratory

using fed-batch cultivation (Lee, 1996b). Similarly, recovery methods for PHAs of various

purities from microorganisms have received attention (Tamer et al., 1998a,b).

Wider use of PHAs is prevented mainly by their high production cost compared with the

oil-derived plastics (Byrom, 1987; Choi and Lee, 1997). With the aim of commercializing

PHA, a substantial effort has been devoted to reducing the production cost through the

development of bacterial strains and more efficient fermentation/recovery processes (Lee,

1996b; Tamer et al., 1998a,b; Grothe et al., 1999). From the literature, the major cost in the

PHA production is the cost of the substrate (Yamane, 1992; Yamane et al., 1993). The effects

of substrate cost on the cost of PHA are summarized in Table 1 (Lee, 1996b; Madison and

Huisman, 1999). The yields of PHA from the various substrates are similar (Table 1), with

one exception. Consequently, the price of substrate has the largest influence on the cost of

production of PHA. The cheapest substrate (Table 1) costs $0.22 kg� 1 of PHA compared

with the cost of polypropylene of $0.185 kg� 1 (Kothuis and Schelleman, 1998).

Productivity also has an effect on the production costs. However, this is relative to the

substrate, and downstream processing apparently has a weak effect on the final cost. When

the PHB productivity increased from 1.98 to 3.2 g�l� 1 h� 1, the PHB production cost

Table 1

Effect of substrate cost and PHB yield on the production cost of PHB (Lee, 1996b)

Substrate Price

(US $ kg� 1)

Yield

(g PHB/g substrate)

Substrate cost

(US $ kg� 1 PHB)

Glucose 0.493 0.38 1.35

Sucrose 0.295 0.40 0.72

Methanol 0.180 0.43 0.42

Acetic acid 0.595 0.38 1.56

Ethanol 0.502 0.50 1.00

Cane molasses 0.220 0.42 0.52

Cheese whey 0.071 0.33 0.22

Hydrolyzed corn starch 0.220 0.185 0.58

Hemicellulose hydrolyzate 0.069 0.20 0.34

H. Salehizadeh, M.C.M. Van Loosdrecht / Biotechnology Advances 22 (2004) 261–279 263

decreased from $5.37 kg� 1 PHB to $4.91 kg� 1 PHB (Lee and Choi, 1998). In a

laboratory fed-batch system using A. latus, the highest reported productivity was 4.94

g�l� 1 h� 1 which would lead to production costs of $2.6 kg� 1 PHB (Lee and Choi, 1998).

PHA content of the produced biomass strongly affects the efficiency of the recovery

process. For example, a relatively low PHB content of 50% results in a high recovery cost

of $4.8 kg� 1 PHB. On the other hand, the recovery cost for a process with 88% PHB

content was only $0.92 kg� 1 PHB (Lee and Choi, 1998). A lower PHB content clearly

results in a high recovery cost. This is mainly due to the use of large amounts of digesting

agents for breaking the cell walls and to the increased cost of waste disposal.

A range of PHAs with 0–24% hydroxyvalerate has been produced under the trade

name of BIOPOL by Zeneca Bio Product and other manufacturers (Table 2) and sold in

the USA (under the trade name of PHBV) (Luzer, 1992), Germany and Japan (Lenz,

1995). However, the PHA production price is far above the market price of conventional

plastics ($16 kg� 1 for BIOPOL against $1 kg� 1 for oil-derived plastics) (Lee, 1996b).

Potentially, the production cost can be lowered by process scale-up, to around $8 kg� 1 at a

production rate of 5000 tons PHA/year (Fritz et al., 1998).

The idea of PHA production using mixed culture arose from a recognition of the PHA’s

role as a metabolic intermediate in microbial processes for wastewater treatment.

Biological wastewater treatment usually occurs under dynamic conditions (Van Loos-

drecht et al., 1997). Activated sludge, a well-known mixed culture, is able to store PHA as

Table 2

Manufacturer, microorganisms and raw materials used for the production of biodegradable plastics (Reddy et al.,

2003)

Microorganism/raw material Manufacturer

Alcaligenes eutrophus ZENECA Bio Product, UK

Alcaligenes latus Biotechnologische Forschungs gesellschaft, Austria

Recombinant Escherichia coli starch Bioventures Alberta, Canada, Warner’s Lambert, USA;

Fertec, Novamont Novara, Italy; Biotech Emmerich, BASF,

Ludwigshafen, Bayer Wolf WalsordeLeverkusen, Germany

Cheap substrates Plyferm, Canada

Bacteria Biocrop, USA; Asahi Chemical and Institute of Physical and

Chemical research, Japan


carbon and energy storage material under unsteady conditions arising from an intermittent

feeding regime and variation in the presence of an electron acceptor. The microorganisms

involved experience rapidly changing conditions of availability of nutrients and can adapt

continuously to change in substrate. Microorganisms which are able to quickly store

available substrate and consume the storage to achieve a more balanced growth have a

strong competitive advantage over organisms without the capacity of substrate storage (Van

Loosdrecht et al., 1997). Activated sludge accumulates PHA to around 20% of dry weight

under anaerobic conditions. The PHA content of activated sludge can be increased to 62%

in a microaerophilic–aerobic sludge process (Satoh et al., 1998b; Takabatake et al., 2002).

When compared with a pure culture (more than 88% of cell dry weight) (Lee, 1996b), the

merits of PHA production in open mixed culture would be an enhanced economy, a simpler

process control, no requirement of monoseptic processing, and an improved use of wastes

(Satoh et al., 1998b). A considerable effort has gone in producing PHA using mixed culture

(Ueno et al., 1993; Matsuo 1994; Saito et al., 1995; Hu et al., 1997; Brdjanovic et al., 1998;

Tsunemasa, 1998; Chau and Yu, 1999; Tohyama et al., 2002; Beun et al., 2002; Van

Loosdrecht and Heijinen, 2002; Takabatake et al., 2000, 2002).

This paper reviews the recent trends in the development of PHA production using

mixed microbial cultures. The aspects reviewed include metabolism of PHA production by

mixed cultures; stoichiometry and kinetics of PHB accumulation and conversion; the

effects of carbon source and raw materials on PHA production by mixed cultures; effects

of temperature and process design on PHA production; and the types of PHAs produced

by mixed culture.

2. Metabolism of PHA production by mixed cultures

2.1. PAO/GAO system

PHA is known to play an important role in mixed cultures especially anaerobic–aerobic

processing where electron donor and acceptor availability are separated (Satoh et al.,

1998b). There are two types of microorganisms capable of anaerobic storage of carbon

source in mixed culture (Chech and Hartman, 1990, 1993): (i) the polyphosphate-

accumulating organisms (PAO) and (ii) the glycogen-accumulating organisms (GAO).

PAOs are probably the most widely recognized for producing storage polymers (PHA,

glycogen and polyphosphates). The whole competitive advantage for these organisms is

based on their capacity to utilize the energy stored as poly-P to store exogenous substrate

in the form of PHA when there is no electron acceptor (oxygen or nitrate) available for

energy generation.

GAOs were recognized recently as competitors of PAOs. Effectively, these organisms

rely on substrates which can be fermented (e.g., glucose), and they store the fermentation

products inside the cell rather than excreting them. These organisms can also use internal

stored glycogen for fermentation to PHB. The energy released in the glycolysis process is

subsequently used to accumulate fermentation products (e.g., acetate) in the form of PHB.

PAOs and GAOs proliferate in systems where the substrate is present regularly while an

electron acceptor is absent (Cech and Hartman, 1993).


Both groups of microorganisms can take up acetate (as a model substrate for metabolic

studies) and activate it to acetyl-CoA. Acetyl-CoA is then consumed for the synthesis of

PHB by condensation to acetoacetyl-CoA, reduction to hydroxybutyl-CoA and finally

polymerization to PHB (Fig. 1). Two metabolic models have been proposed for the

enhanced biological phosphate removal process (EBPR). Comeau et al. (1986) and Mino

et al. (1987, 1996) presented models that explained the interaction between phosphorus

release under anaerobic condition and uptake and storage of short-chain fatty acids. The

main difference in their models was the source of electrons for formation of the PHA.

Comeau et al. (1986) proposed the oxidation of substrate in the TCA cycle. Mino et al.

(1998) measured the anaerobic decrease of intracellular carbohydrate and indicated that

the conversion of glycogen to acetyl-CoA delivered the essential reduction of power for

forming the PHA. According to Mino et al. (1996), the anaerobic metabolism of GAOs

resembles that of PAOs (i.e., glycolysis of stored glycogen and substrate conversion to

PHA through either acetyl-CoA or propionyl-CoA and propionyl-CoA production by

succinate-propionate pathway), except there is no involvement of poly-P. PAOs and GAOs

use the conversion of glycogen to PHA in order to produce ATP and NADH. PAOs use

this conversion mainly to produce reducing power, whereas in GAOs, this conversion is

predominantly used for ATP production. GAOs use Embden–Meyerhoff–Parnas pathway

(EMP) (Filipe et al., 2001a) for glycogen hydrolysis, whereas PAOs use the Entner–

Doudoroff (ED) pathway (Maurer et al., 1997; Hesselmann et al., 2000).

The presence and relative proportion of different PHAs is dependent on the type of

carbon substrate available. The different polymers are formed to allow the cells to balance

the redox equivalents produced and needed in conversion of substrate to PHA. Satoh et al.

(1992) demonstrated the formation of other PHAs containing monomeric units HV,

Fig. 1. PHA production metabolism in PAO/GAO system.


3H2MB (3-hydroxy-2-methyl butyrate), and 3H2MV (3-hydroxy-2-methyl valerate).

Satoh et al. (1994) and Liu et al. (1994) explained the existence of HV formation in

polyphosphate-independent anaerobic and aerobic activated sludge. According to HV

fermentation, glycogen is converted to PHA through propionyl-CoA in order to satisfy the

redox balance. Due to the fact that the organisms are subjected to rapid changes between

aerobic and anaerobic conditions, the enzymes of intermediary cell metabolism are still

active under anaerobic condition. This also leads to formation of PHV when acetate is the

sole substrate for PAOs (Pereira et al., 1996).

2.2. Microaerophilic–aerobic system

Although activated sludge acclimatized under anaerobic–aerobic conditions accumu-

lates PHA, there is no guarantee that anaerobic–aerobic operation of the activated sludge

process is best for enrichment of PHA accumulating microorganisms. Ueno et al. (1993)

and Saito et al. (1995) found that sludge accumulated more PHB under aerobic conditions

than under anaerobic conditions. Satoh et al. (1998b) introduced the microaerophilic–

aerobic process where a limited amount of oxygen is supplied to the anaerobic zone of

anaerobic–aerobic operation. In such conditions, microorganisms can take up organic

substrates by obtaining energy through oxidative degradation of some part of the organic

substrates. If the supply of oxygen is sufficient, the microorganism may be able to get

enough energy for assimilative activities such as the production of protein, glycogen and

other cellular components simultaneously with taking up organic substrates. However, if the

supply of oxygen is adequately controlled, the assimilative activity will be suppressed while

letting the microorganism accumulate PHA. By using these conditions, PHA accumulators

are selected regardless of the ability of microorganisms to accumulate poly-P or glycogen,

and the selected PHA accumulators will have a lower tendency to accumulate glycogen.

The aerobic metabolism for PHB formation is shown in Fig. 2. The metabolic model

was formulated by Van Aalst-van Leeuwen et al. (1997) and Beun et al. (2000a,b). The

model includes the following reactions: synthesis of acetyl-CoA from acetate, anabolism

reactions, catabolism reactions, electron transport phosphorylation, synthesis of PHB from

acetyl-CoA, and the synthesis of acetyl-CoA from PHB.

2.3. Feast and famine cycling

Recently, much research has concentrated on the production of PHAs by mixed cultures

when exposed to a transient carbon supply. Activated sludge processes are highly dynamic

with respect to the feed regime. The biomass subjected to successive periods of external

substrate availability (feast period) and no external substrate availability (famine period)

experiences what in the literature is often called an unbalanced growth. Under dynamic

conditions, growth of biomass and storage of polymer occur simultaneously when there is

an excess of external substrate. When all the external substrate is consumed, stored polymer

can be used as carbon and energy source (Fig. 2). In these cases, storage polymers are

formed under conditions that are not limiting for growth. The storage phenomena usually

dominates over growth, but under conditions in which substrate is present continuously for a

long time, physiological adaptation occurs, and growth becomes more important. The

Fig. 2. PHA production pathways in feast/famine conditions.


ability to store internal reserves gives to these microorganisms a competitive advantage over

those without this ability, when facing transient substrate supply. Among the mentioned

systems for industrial production of PHAs, the feast and famine approach is the most

promising because of high PHA accumulation. This approach promotes the conversion of

the carbon substrate to PHA and not to glycogen or other intracellular materials.

3. Stoichiometry, kinetics of PHA accumulation and conversion in mixed cultures

For PAOs, stoichiometry, glycogen consumption and PHA accumulation are indepen-

dent of pH over the pH range of 6.5–8.0. The amount of phosphorus released per mole of

acetate taken up (P/HAc ratio) is linearly dependent on pH, because of the additional

energy requirement for acetate transport at higher pH (Filipe et al., 2001b). Filipe et al.

(2001b) showed that for GAOs under anaerobic condition, the amount of glycogen

consumed per unit of PHA accumulated in the cells increased with increasing pH,

indicating that the energy requirement for acetate uptake increased with pH. In addition,

the rate of acetate uptake was strongly affected by the pH and decreased with increasing


pH. This was attributed to the increased energy requirement for acetate transport across the

membrane. This implies that high pH values in anaerobic phase are bad for GAOs,

suggesting that pH may be manipulated to minimize the presence of GAOs in EBPR

(Filipe et al., 2001a,b). The stoichiometric relations of acetate uptake, glycogen consump-

tion and PHA production are different for PAOs and GAOs. The yield coefficient YP/S (C

mol/C mol) depends on the process and the substrate used. For PAOs and GAOs, the YP/Sbased on acetate as the carbon source is 1.21–1.43 and 1.58–1.93 (C mol/C mol),

respectively. Liu et al. (1994) reported that for each C millimole of acetate taken up, 1.25

C mmol glycogen was consumed, and 1.91 C mmol of PHA were accumulated in GAOs.

The stoichiometry modified for the effect of maintenance gave 1.105 C mmol glycogen

consumed and 1.76 C mmol of PHA accumulated during the uptake of 1 C mmol of

acetate (Filipe et al., 2001a; Liu et al., 1994, 1996). Filipe et al. (2001a) showed that for

GAOs, an increase pH from 6.5 to 8.5 caused 0.92–1.09 C mmol glycogen consumption

and 1.68–1.83 C mmol PHA was produced. Smolders et al. (1994) and Filipe et al.

(2001a) showed that 0.5 C mmol glycogen were consumed, and 1.33 C mmol PHA were

produced per 1 C mmol acetate by PAOs.

Stoichiometry and kinetics of PHA metabolism in aerobic sludge have been studied by

several researchers (Stanier et al., 1959; Zevenhuizen and Ebbink, 1974; Van Loosdrecht et

al., 1997; Beccari et al., 1998). Under the successive periods of external substrate availability

(feast phase) and no external substrate availability (famine phase), it appears that in feast

phase, 66–100% of the substrate consumed is used for storage of PHB, and the remainder is

used for growth and maintenance. The growth rates in the feast and famine phases are

similar, but growth in the feast phase is higher relative to the famine phase. Acetate

consumption and PHB production in the feast period both proceed with a zero-order rate in

acetate and PHB concentration, respectively. PHB consumption in the famine phase as a

carbon and energy source can be described kinetically with an nth-order degradation

equation in PHB concentration. Rate of PHB degradation in famine phase is independent

of the electron acceptor present (Grau et al., 1975; Beun et al., 2000a; Beccari et al., 2002b).

Murnleitner et al. (1997) proposed a reaction order with n = 2/3 and the rate constant value of

0.3 for PHB consumption. The best fit for the entire data under aerobic conditions was

obtained with n = 1.33 and k = 0.44 and also with n = 2/3 and k = 0.09 (Beun et al., 2000a).

Under denitrifying conditions in activated sludge cultures, the process of storage and

degradation of PHB is the same for anoxic and aerobic environments. The anoxic specific

acetate uptake rate was three to four times lower than for aerobic degradation of PHB and

could be described by a power law with the order equaling 0.59 and k = 0.07. Storage of

PHB in the feast period can increase the required COD/N ratio for denitrification by 70%

compared to the situation without storage. In a conventional nitrifying/denitrifying system

with 66% of the time under aerobic condition, storage of PHB increases the COD need by

30% (Beun et al., 2000b).

4. Effect of carbon source on PHA production by mixed culture

Selection of a suitable substrate is an important factor for optimizing the PHA

production and affects the PHA content, composition and the polymer properties. Over


40% of total operating expense of PHA production is related to the raw materials, and

more than 70% of this cost is attributed to the carbon source. By using cheap substrate

sources such as agroindustrial wastes (e.g., whey, molasses and palm oil mill effluents),

PHA production may be made economic (Choi and Lee, 1997; Meesters, 1998). Many

waste streams from agriculture are potentially useful substrates. These include cane and

beet molasses, cheese whey, plant oils and hydrolysates of starch (e.g., corn, tapioca),

cellulose and hemicellulose. Because an open-culture system would be used, not all

substrate would be equally suitable. For example, starch and cellulose hydrolysates could

lead to the growth of glycogen-accumulating organisms (Mino et al., 1996). This problem

can be easily overcome by acidification of sugars, starch and cellulose hydrolysates with a

mixture of volatile fatty acids (VFAs) such as acetic, propionic, butyric and others. Such a

mixture is readily converted to PHAs. Oil refinery waste such as cracker condensate and

effluent of a partial wet oxidation unit are available as potential sources of volatile fatty

acids (Meesters, 1998).

Table 3 presents an overview of possible waste streams and their PHA production

capacities. It can be seen that only three waste streams contain enough organics (measured

as the chemical oxygen demand, COD) to produce 3500 tons PHA/year. The concentration

of these streams ranges from 1.9 to 50 kg COD m� 3. PHA production using the largest

stream (the CSM spraying waste) is not useful because of a high substrate dilution and an

availability for only 3 months per year leading to a large and unproductive reactor.

Vegetable, fruit and garden waste (VFG) is a solid waste and needs to be acidified prior to

use. This introduces an extra step in the production process. Since only a small part of the

VFG will solubilize, a large disposal problem will remain. The percolate from the organic

wet fraction (OWF) of household waste is considered the most suitable substrate for PHA

production. It has a very high volatile fatty acid concentration, is available in large

quantities and can be transported easily. The heavy-metal content can be removed by

precipitation and then possibly recycled. There are also several reports on the production

of volatile fatty acids from anaerobically treated palm oil mill effluent (POME) and

Table 3

Overview of waste streams suitable for PHA production (Meesters, 1998)

Substrate source Flow

(m3 h� 1)

Availability

(months/year)

COD

(kg COD/m3)

Capacity

(ton COD/m3)

Production

(ton PHA/year)

Potato starch production

process (AVEBE)

300 12 2.5 6750 2431

Innuline production

process (Cosun)

60 5 14.0 3066 1134

Sugar beets process (CSM) 3750 3 1.9 15,604 5773

Brewery wastewater

(Heineken)

300 12 2.8 7358 2723

Vegetable, fruit and

garden (VAM)

90 12 15 11,774 4356

Household garbage (OWF) 30 12 50 13,333 4933

Overview of waste streams suitable for PHA production (Meesters, 1998).

In calculations, the yield Y is assumed to be 0.37 kg PHA/kg COD.


utilization of these organic acids for the production of PHA (Hassan et al., 1996, 1997a,b;

Nor Aini et al., 1999).

So far, acetate has been used by a majority of researchers as the carbon source for PHA

(Hollender et al., 2002; Smolders et al., 1994; Filipe et al., 2001a,b; Liu et al., 1997; Satoh

et al., 1992; Beccari et al., 1998, 2002; Beun et al., 2000a,b). Although there are a few

studies on the PHA production by mixed cultures using propionate, butyrate, lactate,

succinate, pyruvate, malate, ethanol (Majone et al., 2001; Beccari et al., 2002; Satoh et al.,

1992), glutamate and aspartate (Satoh et al., 1998a) and glucose (Yu, 2001; Hollender et

al., 2002), but no studies on the optimization of nutrient removal and PHA production by

mixed cultures are available.

The effect of acetate, acetate and glucose and glucose alone on PHA production under

anaerobic–aerobic conditions in a sequential batch reactor (SBR) was evaluated by

Hollender et al. (2002). A rapid and complete consumption of glucose occurred in the

anaerobic period, whereas acetate consumption was slow and incomplete. The produced

PHBV included 17% and 82% HV for acetate and glucose substrates, respectively. The

maximum amount of phosphate release (27 mg P l� 1) and PHA storage (20 mg C g� 1)

during anaerobic phase as well as the highest phosphate (poly-P) (8 mg P g� 1 dry

material) and glycogen storage (17 mg C g� 1 dry material) during aerobic phase were

obtained using acetate. Lemos et al. (1998) demonstrated that acetate uptake by PAOs

leads to the production of a copolymer of hydroxybutyrate (HB) and hydroxyvalerate

(HV), with the HB units being dominant (69–100% HB; 0–31% HV). With propionate,

HV units are mainly incorporated in the polymer. The yield of polymer (YP/S) was found to

diminish from acetate (0.97) to propionate (0.61) to butyrate (0.21). Using a mixture of

acetate, propionate and butyrate, the PHA synthesized was enriched in HV units (Satoh et

al., 1994; Lemos et al., 1998). In this case, the polymer was composed of 2–28% HB and

45–72% HV. Beside HB/HV copolymers, the 3H2MV and 3H2MB have been produced

using lactate, malate, succinate, pyruvate, propionate and aspartate as substrates. A

polymer containing aminobutyrate and an unknown amino acid was produced by EBPR

activated sludge using glutamate under anaerobic conditions. In presence of aspartate, a

new PHA containing 14% HB, 13% 3H2MB, 48% 3HV and 25% 3H2MV was obtained

(Satoh et al., 1998a).

Sawayama et al. (1999) reported the PHB production rate based on acetate media and

PHB content based on dry biomass of about 6.6–14 mg�l� 1 day� 1 and 15.1–25.3%,

respectively, using photosynthetic bacteria in anaerobic activated sludge. The results

showed that PHB was stored in the presence of ethanol but not in presence of glutamic

acid (Beccari et al., 2002). Hu et al. (1997) synthesized a PHBV using butyric acid as the

sole carbon source and suggested that the HV content of the PHBV could reach 54% when

valeric acid was provided in a high concentration. The total organic carbon (TOC) removal

efficiency decreased from 98% to 65% when the concentration of valeric acid in the

medium increased. The PHA content decreased from 40% to 18% when valeric acid was

used as the sole carbon source. There are several reports (Hassan et al., 1996, 1997a,b; Nor

Aini et al., 1999) on the production of organic acids from anaerobically treated palm oil

mill effluent (POME) and utilization of these acids for PHA production. Hassan et al.

(1996) reported the use of POME containing concentrated 100 g�l� 1 organic acids as

substrate for fed-batch PHA production.


Recently, a new method for PHA production was reported that involved passing

nitrogen-deficient paper and pulp wastewater through an aerobic treatment system. The

dissolved oxygen content was kept at stable levels of 14% and 5% in the first and second

stages of the activated sludge plant, respectively. In both stages, the mean total production

of PHA was 41 g�kg� 1, but the PHA from the first stage had a HB/HV ratio of 79:21,

whereas for the second stage, the ratio was 55:45 (Asrar et al., 1999).

5. Effect of temperature

The influence of temperature on the accumulation of PHB in sequencing batch reactor

cultures fed with acetate has been studied by Krishna et al. (1999) under conditions when

the acetate was rate limiting. The PHB formation rate was shown to decrease with

increasing temperature because of an increase of the anabolic rate at higher temperatures.

6. Process design

Table 4 presents a comparison between the different processes of PHA production by

mixed cultures and pure cultures. The sequencing batch reactor (SBR) is the most important

process among the various potential processes for industrial PHA production. The SBR

technology was developed to gain control over structure and functions of a heterogeneously

composed microbial community in a multipurpose bioreactor exposed to a varying feeding

regime. A SBR is operated under intermittent substrate feeding in order to favor the storage

of the polymer by the sludge. The substrate is fed over a short period of time, followed by a

longer period of a lack of substrate. SBRs are ideal for selection of a robust population with

an elevated ability for PHA production because the biomass grows under transient

conditions (Irvine et al., 1971). This kind of reactor is easy to control and highly flexible,

allowing for a quick modification of the process conditions (i.e., the length of feeding and

the cycle time). The feast/famine process shows YP/S of about 0.24–0.9 (C mol/C mol).

Under aerobic conditions in an SBR, the ratio of acetate uptake to PHB storage is

approximately 0.6 C mol/C mol for systems with a sludge residence time of >2 days which

is comparable to the values (0.7–0.9 C mol/C mol) found by others (Dirks et al., 2001;

Filipe et al., 2001a,b; Carta et al., 2001). The ratio of acetate uptake to PHA storage is

about 0.4–0.5 C mol/C mol under anoxic conditions (Beun et al., 2002). In the feast and

famine regime, the electron acceptor used for carbon oxidation can be either oxygen or

nitrate. The results indicate that the specific PHA production rate is apparently higher for

the aerobic process compared to anoxic conditions.

If a continuous system for producing PHA is desired, then a possible configuration

could include two reactors in series in which a plug flow reactor (PFR) is followed by a

continuous stirred tank reactor CSTR, coupled to a settler or membrane filter. The

concentrated biomass is recirculated to the PFR. The effluent exiting from the PFR would

contain biomass with a maximum PHA content. This effluent would be divided in two

streams, one flowing directly to the CSTR and the other stream flowing to a sludge

concentration step (membrane or settler). The concentrated sludge would be harvested for

Table 4

Comparison of different processes used for PHA production by mixed cultures

Carbon source PHAa

composition

Yieldb

( YP/S)

QPc PHA%d YG/Se Reference

PAOs (anaerobic/aerobic process)

Acetate 90:10:0:0 1.33 0.166 16.7 0.50 Smolders et al., 1994

VFAsf 62:38:0:0 1.71 0.089 23.0 1.12 Levantesi et al., 2002

Acetate 100:0:0:0 0.80 – – 0.33 Bond et al., 1999

Acetate 88:12:0:0 1.37 – – 0.45 Liu et al., 1997

Acetate 89:11:0:0 1.33 0.248 – 0.51 Hesselmann et al.,

2000

Acetate 69:31:0:0 1.43 0.020 2.6 1.06 Satoh et al., 1992

Propionate 7:59:0:34 1.85 0.020 2.5 0.80 Satoh et al., 1992

Malate 21:75:0:4 1.50 0.004 0.9 0.83 Satoh et al., 1992

Lactate 21:76:0:3 1.25 0.006 1.8 0.67 Satoh et al., 1992

Pyruvate 46:54:0:0 0.67 0.008 1.1 0.50 Satoh et al., 1992

Succinate 8:74:0:18 0.93 0.007 1.6 0.53 Satoh et al., 1992

Acetate 75:25:0:0 1.21 0.047 20.1 0.41 Lemos et al., 1998

Propionate 28:72:0:0 0.81 0.009 12.8 1.34 Lemos et al., 1998

Butyrate 60:40:0:0 0.27 0.004 13.1 0.51 Lemos et al., 1998

VFAsf 55:45:0:0 0.77 0.023 12.1 0.26 Lemos et al., 1998

Acetate 88:10:2:0 1.34 0.091 16.4 0.60 Satoh et al., 1996

Propionate 2:45:3:50 1.82 0.097 26.7 0.55 Satoh et al., 1996

VFAsg 14:51:5:30 1.68 – 28.3 0.55 Satoh et al., 1996

Lactate 32:53:5:10 1.63 0.007 10.2 0.69 Satoh et al., 1996

Aspartate 14:48:13:25 0.93 – – 0.21 Satoh et al., 1998b

GAOs (anaerobic/aerobic process)

Acetate 67:26:5:2 1.76–1.91 0.060 15.6 1.11–1.25 Liu et al., 1994

Acetate 89:11:0:0 1.68–1.83 0.006–0.025 19.9 0.92–1.09 Filipe et al., 2001a

Acetate 63:27:7:3 1.93 – – 1.38 Satoh et al., 1994

Propionate 6:59:8:27 1.67 – – 1.00 Satoh et al., 1994

Acetate 69:31:0:0 1.58 – – 1.43 Bond et al., 1999

Feast/famine process (aerobic)

Acetate 100:0:0:0 0.60 0.46 31.0 – Beccari et al., 1998

VFAsh 50:50:0:0 0.45 0.33 62.0 – Beccari et al., 1998

Acetate 100:0:0:0 0.41–0.62 0.23–0.27 12.0 – Beun et al., 2000a

Acetatei 100:0:0:0 0.60–0.70 0.54–0.66 – – Beun et al., 2002

Acetate 100:0:0:0 0.61–0.65 0.025–0.039 – 0.031–0.13 Beun et al., 2002

Acetate 100:0:0:0 0.40 0.011 – – Beccari et al., 2002

Ethanol 100:0:0:0 0.33 0.0026 – – Beccari et al., 2002

Acetate 100:0:0:0 0.41–0.61 1.71–5.73 66.8 – Dionisi et al., 2001b

HAc + Gluj 100:0:0:0 0.60–0.90 0.17–0.28 – – Carta et al., 2001

Feast/Famine process (Anoxic/Aerobic)

Acetate 100:0:0:0 0.40 0.063 16.1 – Beun et al., 2000a,b

Acetate 100:0:0:0 0.24–0.49 1.71 – – Dionisi et al., 2001a

Acetate 100:0:0:0 0.27 0.032 – – Majone et al., 2001

Ethanol 100:0:0:0 0.45 0.021 – – Majone et al., 2001


Table 4 (continued)

Carbon source PHAa

composition

Yieldb

( YP/S)

QPc PHA%d YG/Se Reference

Microaerophilic–aerobic process

Acetate 100:0:0:0 – 0.063 62.0 – Satoh et al., 1998b

Pure culture

(Glucose)

A. eutrophus

100:0:0:0 0.42 0.013 76 – Kim et al., 1994

a PHA% (HB/HV/HMB/HMV).b C mol HA/C mol substrate.c C mol HA/C mol biomass.d g polymer/g biomass.e C mol glycogen/C mol biomass.f Mixture of acetate, propionate and butyrate.g Mixture acetate and propionate.h Mixture of acetate, propionate, butyrate and valerate.i Acetate is very high concentration.j Mixture of acetate and glucose.


PHA extraction. In this case, the plug flow reactor would provide the feast period, and

therefore, its hydraulic retention time should be determined by the time required for the

complete exhaustion of the substrate. The CSTR mimics the famine period, and its

hydraulic retention time should be defined by the time needed to starve the sludge.

Beccari et al. (1998) used a CSTR with intermittent feeding. This process selects for,

and produces sludge with, a high storage capacity. The PHB storage yield ranged from

0.06 to 0.5 g COD/g COD, and the sludge accumulated up to 40–50% of the total dry

weight as PHB (Beccari et al., 1998). Meesters (1998) reported a potential new two-

stage process for PHA production by mixed cultures and compared it to conventional

processes. A detailed process design and economic analysis showed that it was possible

to produce PHA for less than $4 kg� 1 compared to $8 kg� 1 for pure culture. This cost

reduction was achieved because of low substrate costs (85% lower) and capital costs for

investment (50% lower) (Liu et al., 1997). Compared with the conventional PHA

production process, this process was more environmentally friendly. Table 5 shows that

Table 5

Comparison of emissions for PHA production by mixed cultures, pure culture and the production of

polypropylene (PP)

PHA (mixed culture)

(kg/ton)

PHA (pure culture)

(kg/ton)

Polypropylene

(kg/ton)

Chlorinated compounds < 20 110 0.24

Heavy metals 0 0.7 5.77

N compounds to wastewater 10 364 0.4

Other emissions to water 5.24 5.24 0.9

CO2 to air 3000 8920 4257

Energy used (GJ) 39 99.7 6.2

All emissions include production of raw materials (Kothuis and Schelleman, 1996).


the new process was cleaner than earlier processes. The mentioned process did not need

monoseptic operation and could save 61 MJ of energy per kilogram of PHA (Meesters,

1998).

7. Types of PHA produced by mixed culture

Although the physical and chemical characteristics of PHAs obtained by pure

cultures have been widely studied (Doi, 1990), the knowledge of PHAs synthesized

by mixed cultures is scarce. The homopolymer PHB is highly crystalline (55–80%

crystalline) and therefore has a low impact strength and resistance to brittle failure. The

glass transition temperature and the melting point of PHB are approximately 5 and 175

jC, respectively (Lee, 1996a). The industrial copolymers, poly(3HB-co-3HV), possess

improved mechanical properties due to an increase in impact strength, toughness and

flexibility, caused by the incorporation of HV units in the polymer chains. The

physicochemical characteristics of these copolymers are strongly dependent on the

HV content, and the melting temperature decreases significantly with an increase of the

HV fraction in the copolymer. The incorporation of HV does not affect the degradation

temperature, thus allowing ease of processing. The mechanical properties are also

strongly correlated with the average molecular weight of the polymer. The polymer with

a longer chain length is more resistant to mechanical forces. The incorporation of

different monomers other than HV and a longer chain length tends to decrease further

the crystallinity and, consequently, the glass transition temperature (Braunegg et al.,

1998; Doi, 1990).

Table 6 shows some physicochemical characteristics of bacterial polyesters obtained

from mixed and pure cultures. The results indicate that the polymers obtained by mixed

cultures bear similar average molecular weights, polydispersities and crystallinity as those

obtained using pure cultures.

Table 6

Comparison between PHA properties obtained by mixed cultures and pure cultures (Serafim et al., 2001;

Matasuski et al., 2000)

PHA HB/HV mole

ratio (%)

MW

(� 10� 5)

MW/Mn Tg(jC)

Tm(jC)

DHm

(J/g)

Crystallinity

(%)

Mixed cultures

m1 75:25 6.5 1.9

m2 28:72 6 2.1

m3 60:40 4 1.6

Pure cultures

P1 100 12 1.8 4 178 91 69

P2 94:6 13 2.3 � 8 133 39 30

P3 88:12 4.9 1.4 � 13 106 38 29

P4 3:97 3.7 2.3 � 44 42 10 8

m1, m2 and m3 were fed with acetic acid, propionic acid and butyric acid using SBR (EBPR), respectively.

P1, P2, P3 and P4 were produced by recombinant strains of Pseudomonas sp. 61-3.


8. Conclusions

Cost effectiveness is one of the main factors that has prevented the use of PHAs as a

biodegradable commodity plastic. Broader use of PHA in packaging and disposable

products as a potential solution to a significant environmental problem depends heavily on

further reducing the cost and establishing a novel PHA production strategy. The use of

waste material as substrates for PHA production can lead to reduced production costs and

a reduced environmental impact of the PHA production. The use of mixed microbial

cultures facilitates the use of mixed substrates. Cost for these processes can be sustainably

reduced because cheap substrates and nonsterile reactors are used and little process control

is needed. The inherent culture stability and use of open fermentation eliminate the

traditional bottlenecks of continuous monoseptic fermentations.

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