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Polyhydroxyalkanoates production from waste biomass

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2016 IOP Conf. Ser.: Earth Environ. Sci. 36 012040

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International Conference on Chemical Engineering and Bioprocess Engineering IOP Publishing
IOP Conf. Series: Earth and Environmental Science 36 (2016) 012040 doi:10.1088/1755-1315/36/1/012040

Polyhydroxyalkanoates production from waste biomass

A K H Nor Aslan1, M D Mohd Ali1, N A Morad1 and P Tamunaidu1*

1
Shizen Conversion & Separation Technology (SHIZEN) iKohza, Malaysia-Japan
International Institute of Technology, Universiti Teknologi Malaysia (UTM) Kuala
Lumpur, Malaysia

E-mail: 1pramila@utm.my

Abstract. Polyhydroxyalkanoates (PHAs) is a group of biopolymers that are extensively

researched for such purpose due to the biocompatibility with mammal tissue and similar
properties with conventional plastic. However, commercialization of PHA is impended by its
high total production cost, which half of it are from the cost of pure carbon source feedstock.
Thus, cheap and sustainable feedstocks are preferred where waste materials from various
industries are looked into. This paper will highlight recent studies done on PHA production by
utilizing crop and agro waste material and review its potential as alternative feedstock.

1. Introduction
Polyhydroxyalkanoate (PHA) is one of the most researched bioplastic in hope to replace conventional
plastic of today. This biopolymer is a group of naturally-occuring biopolyesters produced by
microorganisms under imbalanced growth condition due to carbon source surplus and/or limitation of
essential nutrient such as phosphorus and nitrogen [1]. Classification of PHAs depends on the number
of carbon atoms present in their monomers as short-chain-length PHAs (scl-PHA; three to five C-
atoms), medium-chain-length PHAs (mcl-PHA; with six or fourteen C-atoms) and long-chain-length
PHAs (lcl-PHA; more than 14 carbon atoms) [1].
Currently, PHA and its co-polymers have been researched for its potential applications in various
industry and fields, for example, poly-(3-hydroxybutyrate) (P3HB) is a homopolymer of PHAs group
that are toxicologically safe and exhibit high biocompatibility with mammalian cells, making them
suitable for medical applications and food industry [1,2,3]. Additionally, the thermoplastic properties
of P3HB and its biodegradability, without generation of toxic by-products, makes it a potential
sustainable alternative to conventional plastics [2,3]. Unfortunately, PHA production cost remains
substantially expensive (about USD8–11 per kg) compared to other polymers, and has been one of the
main obstacle in exploitation of PHA as commercial commodity [2].
Most of the carbon sources used for conventional PHA production are of pure raw material,
comprising of pure carbohydrates (glucose, sucrose, maltose, starch), fatty acids and its derivatives,
methanol and alkanes [2]. In terms of the production cost, carbon source for microbial growth
attributed to 50% of the total costs [3], thus it is desirable to find cheap alternative carbon feedstocks
in order to improve sustainability of PHA production while obtaining commercial viability through

1
To whom any correspondence should be addressed.
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International Conference on Chemical Engineering and Bioprocess Engineering IOP Publishing
IOP Conf. Series: Earth and Environmental Science 36 (2016) 012040 doi:10.1088/1755-1315/36/1/012040

fermentation. As such, inexpensive carbon sources such as agro-industrial by-products and

agricultural residues have been reported as potential alternative substrates.
Therefore, this paper will review on the current advances involving utilization of biomass from
various waste from agriculture, agro-industrial practice and domestic waste for PHA production as
well as the challenges and opportunities for commercialisation.

2. PHA Production from Biomass

2.1. Sugar Rich Feedstock

Sugar producing plant such as maple tree, which is known for its sweet sugar sap, has been studied as
carbon feedstock for PHA production. Yezza et al. employed maple sap a carbon source to
Alcaligenes latus (now known as Azohydromonas lata) for the production of poly-beta-
hydroxybutyrate (PHB) and was able to produce up to 78% of this bioplastic monomer [4]. However
when feedstock is varied, for example conversion of sugarbeet juice using the same microbe A. lata,
Wang et al. reported lower PHA yield of 66% compared to pure sucrose which gave 80.0% of PHA
[5]. This suggests that PHA yield depends on the total chemical composition of the sap/juices used. In
another study, Suwannasing et al. utilized juice from pineapple for the production of PHB using
isolated Bacillus sp. SV13 from agriculture soil. The study showed that 56% of PHB could be
produced from pineapple juice compared to pure glucose and sucrose (18.5% and 29%) [6].
In a relatively new sugar rich feedstock, Zahari et al. showed that oil palm frond can be simply
compressed to obtain their plant juice or sap, which contains high glucose of 53.95 gl -1 that constitute
about 70% of its total sugar content [7]. The study reported a PHA yield of 30.5 g/l, comprising of 75
wt% of the biomass dry weight.
Besides virgin feedstocks, sugar rich by-products are also readily available as raw material for
bioconversion into high value products. Molasses is a by-product of sugar manufacturing industries
and is currently being utilized as a carbon source for industrial scale fermentations due to its
abundance and low price. It is estimated that 12 million ton of beet molasses and 39 million ton of
cane molasses are readily available for further conversion [4]. In a study by Albuquerque et al., a
three-step fermentation strategy was proposed for cane molasses fermentation to produce PHA. The
molasses were fermented to organic acids in the first step followed by PHA accumulation, and then
finally, batch fermentation for the production of PHA, where about 30% cell dry weight (CDW) of
PHA is produced. [8]. Similarly, soy molasses has also shown positive conversion to PHA resulting in
5-17% of mcl-PHA production using Pseudomonas corrugata [9]. While prior studies shows low yield
of PHA in molasses feedstock, a recent study done by Akaraonye et al. for example has claimed able
to produce 61.07% yield of PHA in Bacillus cereus SPV. They claimed that by limiting the oxygen
supply in their media, this lead to anaerobic condition for growth which may have led to enhanced
yield of PHA [10].

2.2. Waste Lipids and Industrial Wastewaters

Cooking oils, specifically waste cooking oils have been considered as potential alternative carbon
feedstock for PHA production. Triacylglycerides (TAGS) from the fatty acids of the waste oil is seen
as valuable alternative feedstock for the production of mcl-PHAs and lcl-PHAs [11]. A study done by
Obruca et al. using waste rapeseed oil with precursor propanol is able to produce up to 76% PHA and
8% 3-hydroxyvalerate (3-HV) in co-polymer [12]. Another study using same carbon source showed
that the high nitrogen content in heated oil due to thermal degradation was able to increase cell growth
and eventually increase PHA yield [13].
Crude glycerol gained more interest as feedstock due to its large quantities as by-product in various
industrial processes, particularly in biodiesel production. Crude glycerol from soy-based biodiesel has
been studied by utilizing Pseudomonas corrugata 388 and Pseudomonas oleovorans NRRL B-14682
resulting in accumulation of mcl-PHAs [14]. A study by Mozumder et al. yielded the highest PHA
content up to 63% by using crude glycerol via a three step fermentation process [15]. In an attempt to

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International Conference on Chemical Engineering and Bioprocess Engineering IOP Publishing
IOP Conf. Series: Earth and Environmental Science 36 (2016) 012040 doi:10.1088/1755-1315/36/1/012040

reduce production cost further, a study utilizing crude glycerol combined with meat and bone meals as
a nitrogen source was done and it resulted in 5.9 gl-1 PHAs and a co-polymer PHBV without addition
of precursor in the media [16].
Another interesting alternative carbon source is wastewater from industrial practices such as
biodiesel wastewater, food processing waste effluent, municipal wastewater and brewery waste
effluent [17]. In the first step, organic carbon sources from wastewater are converted into volatile fatty
acid in aerobic activated sludge, followed by PHAs conversion by mixing cell cultures in the second
step. Generally, PHA concentrations from wastewater are low in current studied settings, with only
around 50% production of PHA [17, 18]. Martinez et al. managed to accumulate 55% PHA content
with 11% of HV when pure Cupriavidus necator is fed with pre-treated olive mill wastewater, without
synthetic precursor under acidogenic condition [19]. However, another study by Khardenavis et al.
reported high PHA yield of 67% from mixed microbial culture (MMC) by using rice grain distillery
wastewater as carbon source [20]. The high yields were only achieved after adding di-ammonium
hydrogen phosphate (DAHP) as extra nitrogen source.

Figure 1. Various feedstock and their respective pre-processing technology prior to microbial PHA

2.3. Lignocellulosic Biomass

Agricultural practices often produces abundant lignocellulosic biomass. Utilization of non-food
lignocellulosic materials for PHA production is preferable over food crops. Usually, lignocellulosic
materials need to be pre-treated and undergo hydrolysis to convert to sugar monomers. In a study by
Cesario et al., PHA production from wheat straw hydrolysate is able to accumulate up to 72% CDW
through fed-batch fermentation using Burkholderia sacchari DSM17165 [21]. It was also stated that
poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV production by utilizing the same carbon source
can also be achieved using propionic acid as a co-substrate. Most research on utilizing lignocellulose
as carbon feedstock centre around the conversion of the monomer sugars from hemicelluloses.
Other agro industrial residues such as inexpensive mixture of extruded rice bran and wheat bran
also has been studied by using haloarchaeon Haloferax mediterranei, which resulted in 56% of PHA
content [22]. While the resulting PHA is still relatively low, it is important to note that extraction
using archaea is inexpensive and safe due to the lack of chemicals used for the separation and
purification of the PHA accumulated in the haloarchaea species.

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International Conference on Chemical Engineering and Bioprocess Engineering IOP Publishing
IOP Conf. Series: Earth and Environmental Science 36 (2016) 012040 doi:10.1088/1755-1315/36/1/012040

Therefore, the production of PHA depends on many factors, which is the feedstock quality,
microbes used, nutrients and pre-processing technology. The PHA production via microbial
fermentation of various feedstocks and their pre-processing technology is summarized in figure 1 and
table 1 below.
Table 1. PHA production in term of cell dry weight percentage (%CDW) from microbial
fermentation of various waste biomass

Biomass Microorganism PHA Content, Cell Dry Ref.

Weight (%CDW)

Soy molasses Pseudomonas corrugata. 5.00-17.00 [9]

Molasses (sugarcane) Bacillus cereus SPV 61.07 [10]
Crude glycerol Cupriavidus necator DSM545 62.70 [15]
Wheat straw Burkholderia sacchari DSM 72.00 [21]
hydrolysate 17165
Extruded rice bran Haloferax mediterranei 56.00 [22]
and wheat bran
Rice grain distillery Activated sludge (MMC) 67.00 [20]
wastewater with
nutrient added
Olive Mill Cupriavidus necator DSM 55.00 (11% 3HV [19]
Wastewater 545
co-polymer)
Waste rapeseed oil Cupriavidus necator H16 76.00 (8% 3-HV [12]
co-polymer)
Pineapple juice Bacillus sp. 48.89 [6]
Sugarbeet Juice Azohydromonas lata 65.60 [5]
Maple Sap Azohydromonas lata 77.60 [4]
Oil Palm Frond Juice Cupriavidus necator NCIMB 75.00 [7]
11599

3. Challenges and Opportunities

Cellulose and hemicellulose from lignocellulosic materials are both excellent non-food carbon source
to be used in different biological processes after hydrolysis to monomeric sugars. However, the
process is cost inefficient due to natural lignin component that is recalcitrant to degradation. Various
pre-treatments are required to loosen up the lignocellulose structure followed by utilization of
enzymes or special microbial strains to convert the holocellulose into sugar feedstock which
contributed to additional cost [18]. Furthermore, hydrolysis of lignocellulose waste by chemical and/or
enzymatic treatments unfortunately releases several inhibitory compounds, such as phenolic, furans
and organic acids which can have negative effect to microbial fermentation. To solve this, an adapted

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International Conference on Chemical Engineering and Bioprocess Engineering IOP Publishing
IOP Conf. Series: Earth and Environmental Science 36 (2016) 012040 doi:10.1088/1755-1315/36/1/012040

tolerant strain of C.s necator is engineered, which can grow in diluted bagasse hydrolysates and
efficiently reduce acetic acid, formic acid, furfural and acid soluble lignin while producing PHAs [23].
The polyesters produced were mainly PHB (57% on CDW), but also included PHBV. C. necator
strain mainly utilizes glucose as carbon source leaving potential down streaming of the unutilised
sugar components in the treated hydrolysates in the production of biofuels and other chemical.
As for the usage of wastewater for PHA production, it inevitably causes the formation of non-
storing PHA population which may adversely affect production of the biopolymer. In a study by
Marang et al. using a mixed culture dominated by Methylobacillus flagellatus, an obligate
methylotroph, that cannot store PHA, and Plasticicumulans acidivorans, a known PHA producer, has
lowered the PHA content from 80 to 66% wt, due to the presence of the non-storing population [25].
The fermentation was enriched with acetate and methanol, as they reduced the effect of methylotroph
M. flagellatus on the PHA production [25].
Usage of virgin oils such as soybean produces higher PHA compared to waste oils, which may
have been due to the presence of impurities in the waste oils [18]. However, the usage of edible oils
for PHAs production is no longer considered cost effective due to increase in price. Thus the focus is
on non-edible oils and waste cooking oils. Crude glycerol from biodiesel is one of the feedstock that
cannot be used as raw material for drug, food and pharmaceutical industries due to the presence of
major impurities [15]. Refining crude glycerol is costly, thus the biological conversion of crude
glycerol to higher value chemicals, such as PHA, is more desirable. Unfortunately, it is important to
note that utilization of glycerol can affect the quality of the polymer by reducing its molecular mass,
thus changing the properties of the PHA [15].
Conversely, PHA production using sugar based crops generate higher yield, depending on the sap
and strain type used as shown in Table 1. Plant sap or juice consists of high sugar content and as well
as minerals such as potassium, nitrogen and other which are essential for PHA accumulation and
microbial growth [5, 4, 7]. The surplus of sugar and presence of mineral make the plant juice or sap as
potential natural media for microbial fermentation. This is supported by a study by Zahari et al. which
showed that the PHA extracted from oil palm frond juice produced relatively high molecular mass; an
important factor in determining PHA’s physicochemical and mechanical properties [7, 25]. The only
drawback from using plant saps and juices as carbon feedstock is the negative view on using potential
sugar crops for food.

4. Conclusion
PHA differs from conventional plastics as it is non-toxic and biodegradable where the end degradation
products are mostly carbon dioxide and water. Current PHA production is still hindered by raw
material as carbon feedstock and downstream-processing cost. Various waste biomass from agro-
industry and municipal wastes have been looked into as cheap alternative carbon source. However, it
has been highlighted in this paper that each alternative feedstock has its challenges towards
commercialization of PHA. However, sugar based carbon source produced notably higher PHA in
term of CDW, compared to other feedstock and not requiring pre-treatment prior to bioconversion of
PHA. The production of PHA may turn to be cost efficient when alternative or underutilized waste or
crops are used to make non-edible sugars for PHA fermentation. More study on alternative sugar
resources are needed to investigate this notion.

5. References

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International Conference on Chemical Engineering and Bioprocess Engineering IOP Publishing
IOP Conf. Series: Earth and Environmental Science 36 (2016) 012040 doi:10.1088/1755-1315/36/1/012040

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Acknowledgement
The authors would like to acknowledge Universiti Teknologi Malaysia (UTM) for providing Potential
Academic Staff grant (Vote No. 01K52). Additionally, MyBRAIN15 from Ministry of Higher
Education Malaysia as well as Malaysia-Japan International Institute of Technology (MJIIT) for the
provision of research support, scholarship and financial assistance to the student. Supports of all
sources are greatly appreciated.