Plant oils as promising substrates for polyhydroxyalkanoates production

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Journal of Cleaner Production xxx (2014) 1e14

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Review

Plant oils as promising substrates for polyhydroxyalkanoatesproduction

Slawomir Ciesielski a, *, Justyna Mo _zejko b, Nipon Pisutpaisal c

a Department of Environmental Biotechnology, Faculty of Environmental Sciences, University of Warmia and Mazury in Olsztyn, Sloneczna 45G,10-719 Olsztyn, Polandb Department of Microbiology, Faculty of Biology and Biotechnology, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Polandc Department of Agro-Industrial, Food and Environmental Technology, Faculty of Applied Science, King Mongkut's University of Technology North Bangkok,Bangkok, 10800, Thailand

a r t i c l e i n f o

Article history:Received 1 February 2014Received in revised form31 July 2014Accepted 6 September 2014Available online xxx

Keywords:Agricultural feedstockBiopolymersFatty acidsPlant oilsPolyhydroxyalkanoates

* Corresponding author. Tel.: þ48 89 5234144; fax:E-mail address: [email protected] (S. Ciesielski)

http://dx.doi.org/10.1016/j.jclepro.2014.09.0400959-6526/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Ciesielski, SProduction (2014), http://dx.doi.org/10.1016

a b s t r a c t

The development of processes for the production of biopolymer materials is being stimulated by acombination of factors. These factors include the negative effects of petrochemical-derived plastics onthe global environment, depletion of global fossil fuel supplies, and the growing demands of an ever-increasing population for the products deemed necessary for an affluent modern lifestyle. In partic-ular, polyhydroxyalkanoates have attracted attention as environmentally friendly alternatives to thesynthetic polymers that are commonly used. Polyhydroxyalkanoates are polyesters produced andaccumulated in intracellular granules by many microorganisms. Because they are biodegradable andbiocompatible and can be produced by fermentation of renewable feedstocks, they are consideredattractive substitutes for petroleum-derived polymers. To create bacterial polyesters, crude and wasteplant oils, which can be difficult to dispose of, can be recovered and used as feedstock. This paper givesan overview of the potential for the production of polyhydroxyalkanoates with useful physicochemicalproperties by bacteria grown on renewable resources such as plant oils.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Petroleum-based plastics have excellence physical propertiesthat resulted in their wide application in almost all industries. Theuseful life time of articles made of plastic is usually very short,especially when they are used for packaging. After use they areusually discarded, thus the use of synthetic plastics has led to solidwaste management problems. Indeed, plastics are especially trou-blesome to recycle, because different types of plastic need to beprocessed differently to be reformulated and re-used as raw ma-terial. Even in the most developed countries in Europe, plasticrecycling does not exceed 50% (BIO Intelligence Service, 2013).

In response to plastic waste's problems and harmful effects onthe environment, there has been considerable interest in thedevelopment of materials which could be more biodegradable thanpetrol-based plastics. As a result, polymers such as polylactides,aliphatic polyesters, polysaccharides and polyhydroxyalkanoates

þ48 89 5234131..

., et al., Plant oils as promisin/j.jclepro.2014.09.040

have been taken under consideration as substitutes for conven-tional plastics. Polyhydroxyalkanoates (PHAs) are especiallyattractive because they have the proper physical properties and canbe completely biodegraded under various conditions by a multi-plicity of microorganisms within a period of one year (Andersonand Dawes, 1990).

Due to high PHAs synthesis costs, their production on a largescale is limited, therefore there is a need for the development ofnovel processes using inexpensive carbon sources. Such substratescould be plant oils. Especially, the usage of waste plant oils,generated by the food industry and food service, could be reason-able from the economic and environmental point of view. Theconversion of these residues to new biomaterials is fully an eco-innovative approach.

The aim of this review is to give a background on the poly-hydroxyalkanoates, their properties, applications and to systema-tize recent research focused on the potential use of plant oils intheir production. This review highlights challenges in the PHAstechnological approaches to make these environmentally friendlybiopolymers as competitive as conventional plastics. In this paper,the authors consider the opportunities of the usage of plant oils as a

g substrates for polyhydroxyalkanoates production, Journal of Cleaner

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S. Ciesielski et al. / Journal of Cleaner Production xxx (2014) 1e142

promising utilizable carbon source towards valuable biomaterialsproduction.

2. Methods

This reviewwas designed to summarize recent progresses in thePHAs production field using plant oils. Papers from numerous sci-entific journals, commercial websites were selected and reviewed.The literature search was carried out using the most important andavailable databases, such as Scopus and PubMed. Firstly, specifickeywords were selected: polyhydroxyalkanoates, waste plant oils,biopolymers, food waste, byproducts food industry, agriculturalfeedstock. Then, the abstracts were examined and selected basedon the criteria that were chosen to determine the publicationsrelated to the objective of this paper. In particular, the authorsidentified studies: (1) dealing with PHAs production during bac-terial cultivations on pure and spent form of plant oils; (2)providing fermentations strategies together with the description ofcrucial technological parameters; (3) focusing on material proper-ties of PHAs from oils; (4) addressing future prospects of intro-ducing biopolymers into global market.

3. PHAs structure and metabolism

PHAs are divided into two groups according to the number ofcarbon atoms in the monomer units: short-chain length PHAs (scl-PHAs) with 3e5 carbon atoms in eachmonomer unit, andmedium-chain length PHAs (mcl-PHA) with 6e14 carbon atoms in each unit(Fig. 1). The most commonly produced monomer unit is poly-3-hydroxybutyrate [P(3HB)]. Scl-PHAs with poly(3-hydroxybutyrate-co-3-hydroxyvalerate copolymers [P(3HB-co-3HV)] have mechanical properties that are considered moredesirable than P(3HB) homopolymers; mcl-PHAs are even moredesirable, because of their superior thermo mechanical properties.The type of polymer produced is determined by the strain of bac-teria used.

Although the composition of PHAs was determined byLemoigne in 1927, interest in PHAs only started to increase in 1958.That was the year when Macrae and Wilkinson (1958) observedthat Bacillus megaterium stored a homopolymer especially swiftlywhen the glucose-to-nitrogen source ratio of themediumwas high.This homopolymer turned out to be poly-3-hydroxybutyrate[P(3HB)], nowadays the most commonly known and widelyrecognized polymer. Since 1958, knowledge about PHAs has greatlyincreased. These biopolymers are accumulated by both Gram pos-itive and Gram negative bacteria, cyanobacteria and even Achaea(Chee et al., 2010b). Hundreds of bacterial species that can syn-thesize PHAs have been counted, and the number is still increasing.

Accumulating PHAs is a natural way for bacteria to store carbonand energy when environmental conditions are disturbed. In lab-oratory conditions these polyesters are accumulated when bacte-rial growth is limited by depletion of nitrogen, phosphorous oroxygen and an excess amount of a carbon source is still present(Anderson and Dawes, 1990; Ciesielski et al., 2010).

PHAs are accumulated in intracellular granules inside the cells.In Cupriavidus necator (formerly Ralstonia eutropha), 8-13 granules

Fig. 1. Structure of PHAs.

Please cite this article in press as: Ciesielski, S., et al., Plant oils as promisinProduction (2014), http://dx.doi.org/10.1016/j.jclepro.2014.09.040

per cell with sizes ranging from 0.2 to 0.5 mm were detected(Byrom, 1992). The surface of PHA granules is coated with phos-pholipids and proteins, which play a major role in PHA synthesis,degradation and even the whole process of PHA synthesis regula-tion. These proteins facilitate PHA metabolism, protect the granulefrom coalescence, and separate the hydrophobic polymer from theaqueous cytoplasm. By far the most abundant proteins on thegranule are the phasins which has been shown to cover from 27 to54% of the PHA granules' surface in Cupriavidus necator (Tian et al.,2005).

From a chemical point of view, PHAs are polyesters arecomposed of hydroxycarboxylate monomers. At least 150 differentmonomer constituents of PHA have been found, which possesstraight, branched, saturated, unsaturated and aromatic structures(Steinbüchel, 2001). These monomers are all in the R-configurationbecause of the stereospecificity of the biosynthetic enzymes.Monomers with various functional groups on the chain, such ashalogen, hydroxy-, epoxy-, cyano-, carboxyl- and esterifiedcarboxyl groups, have been discovered in mcl-PHAs (Steinbücheland Valentin, 1995).

The mechanical properties of individual PHAs depend on thecomposition of the monomer units. P(3PHB) is highly crystallinebecause it is a completely stereoregular polyester, with all asym-metric carbon atoms in the R-configuration. The high crystallinity(typically 55e80%) makes it relatively stiff and brittle. The glasstransition temperature (Tg) of P(3HB) lies between 5 and 9 �C, andthe melting point (Tm) ranges from 173 to 180 �C. P(3HB-co-3HV) isanother well-studied PHA, composed of both 3HB and 3HVmonomers. Its mechanical properties are considered more desir-able than P(3HB) monomers because its melting point is muchlower, and it is less crystalline, easier to mold and tougher thanpure P(3HB) (Luzier, 1992). These thermo mechanical propertiescan be widely varied because the composition of P(3HB-co-3HV)can range from 0 to 30 mol% 3HV (Braunegg et al., 1998).

Mcl-PHAs have significantly lower melting point temperaturesthan scl-PHAs; they range from 39 to 61 �C and are stronglydependent on the thermal history of the material. Their glasstransition temperature is usually below room temperature, rangingfrom �43 to �25 �C, and they are about 25% crystalline. Thesecharacteristics make mcl-PHAs more flexible and elastic materialsthan scl-PHAs (Sudesh et al., 2000).

The type of PHA produced depends on themetabolic pathway ofthe particular bacteria being used. Scl-PHAs consisting of P(3HB)homopolymers are generally biosynthesized in a three-step processthat is regulated by 3-ketothiolase (PhaA), acetoacetyl-CoA reduc-tase (PhaB) and the SCL PHA synthase (PhaC). The most commonmetabolic pathway, found in awide range of bacteria producing scl-PHAs, is probably the generation of P(3HB) from acetyl-CoAs. Thispathway has been extensively studied in Cupriavidus necator. In thisbacteria two acetyl-CoA moieties are condensed to yieldacetoacetyl-CoA by 3-ketothiolase (PhaA). The product is subse-quently reduced to (R)-3HB-CoA by an NADPH-dependent acetoa-cetyl-CoA reductase (PhaB). Only (R)-isomers are accepted assubstrates by PHA synthase (PhaC), the polymerizing enzyme(Oeding and Schlegel, 1973).

There are three kinds of known pathways for mcl-PHA synthe-sis, found mostly in Pseudomonas species. Each pathway uses adifferent substrate to create 3HAs, which are then used to syn-thesize mcl-PHAs. All Pseudomonas spp. will only accept 3HAs of6e14 carbon atoms. This is due to the specificity of PHA synthase,the enzyme responsible for polymerization of hydroxyalkanoic acidthioesters (Koller et al., 2010a).

In the first pathway that creates 3HAs for mcl-PHA synthesis,aliphatic carbon sources are degraded via b-oxidation to produce 3-hydroxyacyl-CoA. This b-oxidation intermediate is in the (S) form,


Fig. 2. General scheme of the PHAs production process.

S. Ciesielski et al. / Journal of Cleaner Production xxx (2014) 1e14 3

so it is unusable by PHA synthase. To make it usable, enoyl-CoA isconverted by an (R)-specific enoyl-CoA hydratase, often termedPhaJ, to produce polymerizable (R)-3- hydroxyacyl-CoA. In thesecond pathway, mcl-PHAs are synthesized by fatty acid biosyn-thesis from unrelated substrates such as glucose, gluconate orethanol. These substrates are converted into fatty acids, whichPHA-polymerase uses to synthesize mcl-PHA polymers (Madisonand Huisman, 1999). The third kind of pathway is a chain elonga-tion reaction, in which acetyl-CoA moieties are condensed to 3-hydroxyacyl-CoA (Witholt and Kessler, 1999).

Two PHA synthase genes, phaC1 and phaC2, have been identifiedand characterized in Pseudomonas species (Prieto et al., 1999); theyare separated by the phaZ gene, which encodes PHA depolymerase.The mcl-PHA biosynthesis gene locus includes also phaD, whichencodes a putative transcriptional regulator of the TetR family withan important role in polyhydroxyalkanoates biosynthesis (Klinkeet al., 2000). Finally, the genes phaF and phaI which are tran-scribed in the opposite direction, encode polyhydroxyalkanoategranule associated proteins. PhaF has also been considered as anegative regulator of PHA biosynthesis genes (Prieto et al., 1999).

4. Microorganisms and fermentation strategies

To effectively produce PHAs with microorganisms, several fac-tors must be considered. Although these include the ratio of carbonsource to nitrogen source, the presence of macro- and microele-ments, physical parameters including the period of cultivation, thetemperature of cultivation, and the optimum pH for cultivation (Leeet al., 2004), the most important factor is the PHA producer, whichis usually a species of bacteria. At this time, at least a few hundredbacterial species have been studied that possess the ability toaccumulate PHAs. Most of them, however, can not be regarded aspromising biopolymer producers, because their ability to synthe-size and accumulate PHAs is insufficient.

When consideringmicroorganisms for the industrial productionof PHAs, several factors need to be considered, such as the ability ofthe cell to utilize an inexpensive carbon source and to achieve goodrates of growth and polymer synthesis. Only some of the bacterialspecies that have been studied can accumulate PHAs in satisfactoryamounts and at a low cost. These include Alcaligenes latus, Azoto-bacter vinelandii, B. megaterium, Paracoccus denitrificans, Proto-monas extorquens, Cupriavidus necator and Pseudomonas oleovorans(Chanprateep, 2010).

The general scheme presenting PHAs production process isshown on Fig. 2. Several PHA production strategies have beendescribed, including batch, fed-batch and continuous processes.Each of them can be conducted under a variety of conditions. Batchand fed-batch fermentations are widely used in industrialfermentation processes. For batch cultivation, two approaches havebeen developed: one-stage and two-stage cultivation. In one stagecultivation, microorganisms are used that can simultaneously grow


and accumulate PHAs. Two-stage cultivation is performed whenmicroorganisms need nutrient limitations. The first stage consistsof a cell growth phase which is carried out in a separate nutrient-enriched medium to ensure sufficient biomass. The cells are thentransferred to a nutrient limited medium for the PHA accumulationphase. During this nutrient limitation stage, the cells are unable tomultiply and their number remains almost constant. However, thecells begin to increase in size and weight due to the intracellularaccumulation of PHAs as a storage product (Lee, 1996).

Fed-batch cultivation is more efficient than batch cultivation forachieving high product- and cell-concentration because it ispossible to avoid inhibition by overly high concentrations of thesubstrate (Chee et al., 2010a). Single fed-batch fermentations thatare nitrogen limited lead to low amounts of polymer, because thereis not enough accumulation of biomass (Katircioglu et al., 2003),thus two-stage fed-batch cultivation is usually employed (Dinizet al., 2004).

Continuous cultivation is also recognized as a viable strategy forPHAs production in order to overcome the high sensitivity of theproduction strains to high concentrations of the required sub-strates, which are mainly fatty acids and their derivatives (Kolleret al., 2010a). It has been employed for production of both scl-PHAs (Mothes and Ackermann, 2005) and mcl-PHAs (Jung et al.,2001). However, this type of cultivation is impractical at a largescale, which makes fed-batch fermentation the best method forindustrial PHA production.

In addition to the wild strains that produce PHAs, efforts havebeen made to use genetically recombined strains to produce PHAs.For example, Escherichia coli do not normally produce PHAs, butgenetic engineering has made it possible to introduce changes thatenable the bacteria to produce biopolymers. In E. coli, variousmetabolic pathways have been developed that lead to highly pro-ductive PHAs accumulation at a low-cost (Ahn et al., 2001). Asanother example, in Pseudomonas putida the biosynthetic enzymesthat drive PHA production have been overexpressed. This approachwas described by Kim et al. (2006): two PHA synthases (phaC1 andphaC2) were cloned and overexpressed in P. putida. In bacteria thatheterologously expressed the phaC1 gene, PHA content was 70.5%greater than that of the wild type, when measured as PHA as apercent of dry cell weight. Another approach that has been used inP. putida is inactivation of the gene responsible for PHA depoly-merization (phaZ), which prevents degradation of intracellularPHA. An experiment performed by Cai et al. (2009) showed that aphaZ knockout mutant of P. putida KT2442 accumulated 86% of mcl-PHA of its cell dry weight (CDW), whereas the wild form producedonly 66%.

The use of mixed cultures as bioplastic producers enables moreeconomic production of PHAs than the use of pure cultures. Mixedcultures like activated sludge have a few advantages: the process issimpler to control, monoseptic processing is not required, andcheap substrates and evenwastes can be used as a source of carbon



(Salehizadeh and van Loosdrecht, 2004). This technique imposesselective pressure on highly diverse mixed microbial communities(MMC) to select microorganisms with a certain production capacity(Ciesielski et al., 2008). To enrich the bacterial community withthese bacteria that effectively produce PHAs, selective pressure isapplied in the form of changing periods of presence and absence ofthe external carbon substrate (feast/famine phases) (Serafim et al.,2008).

Established methods of polymer extraction and purificationneed to be made more cost-effective. The simplest method isextraction of PHAs with a solvent, such as chloroform, dichloro-methane, dichloroethane, ethylene carbonate or acetone. The sol-vent first makes the cell membrane more permeable and then itsolves the PHAs. The polymers are then concentrated and precipi-tated with methanol or ethanol (Anderson and Dawes, 1990). Thismethod is costly and environmentally unattractive because it uses alarge amount of solvent, which is difficult to dispose of. Othermethods relying on digestion of non-PHA cell material also havedisadvantages. For example, hypochlorite can degrade the poly-mers (Ryu et al., 2000), and enzyme digestion is too expensive to beeconomically reasonable.

Several methods of polymer extraction and purification havebeen investigated. Mechanical cell-disruption methods like usingbead mills and high pressure homogenization are effective andinexpensive (Jacquel et al., 2008). Supercritical fluid disruption(Hejazi et al., 2003) and dissolved air flotation (van Hee et al., 2006)still need to be further developed.

5. Renewable resources as substrates for PHAs production

Despite the basic attractiveness as a substitute for the syntheticplastic, the major barrier for commercial production and applica-tion of PHAs is the high cost of bacterial fermentation, making PHAsabout 15 times more expensive than the petroleum derived poly-mers such as polypropylene (Singh et al., 2009). The industrialproduction of PHAs is determined by the carbon substrate cost. Theeconomic analysis for PHAs production indicated that carbon sub-strates constitute the significant part of the overall production cost(Braunegg et al., 2004). Thus, researchers are trying to find alter-native resources to enable the production of renewable plastics tobe competitive in the global plastics market.

Renewable biomass can be considered as the best carbon sourcefor large scale production of PHAs. The sources of renewablebiomass generally, originated from industry, agriculture or house-hold waste materials (Table 2). Such materials are mainly producedin agriculture and industrial sectors that are closely related toagriculture (Braunegg et al., 1998; Solaiman et al., 2006; Khannaand Srivastava, 2005; Koller et al., 2010b). Natural feedstocks thathave been used for PHAs production include food and starch-basedwastes, plant oils, organic industrial wastewater and biodiesel in-dustry residuals (Braunegg et al., 2004; Koller et al., 2010a; Hassanet al., 2012). The selection of carbon sources should focus not onlyon market prices but also on continuous availability and globalprice consistency. In addition, inexpensive carbon sources such asagricultural wastes and industrial by-products may incur additionalcosts due to pre-treatment steps, extended cultivation times, andpurification (Chanprateep, 2010).

The selection of the appropriate waste stream as feedstock forbiotechnological purposes mainly depends on the global regionwhere industrial PHAs production will be carried out. The costs ofPHAs production will be lower, if production plant will be inte-grated with the existing production lines, where the feedstock isdirectly accrued as waste streams (Koller et al., 2010a). One of thepromising substrates, which could be converted into poly-hydroxyalkanoates is cheesewhey, a side-product of dairy industry.


Cheese whey contains lactose, lipids and soluble proteins and it isavailable in large amounts in these parts of the world, in whichdairy industry is developed. In many European countries whey issurplus product, thus spending it for PHAs production could be anadded value. Whey was successfully employed for PHAs synthesisby recombinant E. coli K24K (Nikel et al., 2006). Obtained resultsrevealed, that this substrate application can lead to PHAs produc-tion in amount of 70.1 g/L with PHAs concentration on the level of72.9%. Recombinant Cupriavidus necator DSM 545 (phaZ knockout)was able to synthesize P(3HB) when whey permeate and hydro-lyzed whey permeate were used as the carbon source (Povolo et al.,2009). Whey was also used for heteropolymer production usingThermus thermophilus HB8, which accumulated it in concentrationof 35% (Pantazaki et al., 2009).

Starch, one of the most important products synthesized byplants, can also be used as alternative viable substrate for efficientPHAs production. Starch is primarily comprised of two differentglucose polymers, amylose and amylopectin. Amylose whichmakesup 15e25% of starch is comprised of long linear chains of glucoseresidues, linked via a-(1,4)-glycosidic bonds. In contrast, amylo-pectin consists mainly of a-(1,4)-linked glucose units (Kossmannand Lloyd, 2000). Most processes for PHAs production based onstarch require the conversion of starch to easily convertible sub-strates such as glucose by enzymatic or chemical hydrolysis (Kolleret al., 2010b). Huang et al. (2006) working with Halofera medi-terranei obtained a cell concentration of 140 g/L, PHA concentrationof 77.8 g/L and PHAs content of 55.6% of CDW in a repeated fed-batch fermentation in case when the mixture of extruded ricebran and corn starch in a ratio of 1:8 was employed. Recently,P(3HB) was also synthesized by application of hydrolyzed cassavastarch. Krueger et al. (2012) described that B. megaterium accu-mulated this biopolymer at the level of 29.7% of CDW whenmaximum production reached 4.97 g/L. Moreover, other papersdescribe the PHAs production on the base of starch coming fromwaste potato (Haas et al., 2008) and hydrolyzed sago starch (Atifahet al., 2007).

Molasses, a by-product of sugar production, is widely available,inexpensive carbon source for PHAs production. Because, it con-tains nearly 45% sucrose and simple sugars it could be considered asideal substrate for bacterial fermentation. One of the first reports onPHAs production using molasses was written by Page (1992).Author reported, that even 2.5 g/L of P(3HB) can be synthesized byA. vinelandii UWD on a beet molasses-based medium. Accordinglyto Gouda et al. (2001) B. megaterium is able to accumulate up to46.2% of P(3HB) when 3% (w/v) sugar cane molasses was supplied.In other works, genetically recombinant bacteria were alsoemployed to PHA, production using molasses. The authorsdescribed the accumulation by Klebsiella aerogenes of about 3 g/LP(3HB) corresponding to approximately 50% of P(3HB) in cell mass(Zhang et al., 1994). More recently, the ability of recombinant E. colito produce PHAs was investigated by Saranya and Shenbagarathai(2011). In this work the authors showed that the utilized strainallowed production of PHAs at the level of 3.06 g/L, which washigher than the yield obtained by this same strain when sucrosewas supplied (2.5 g/L). The other by-product of sugar cane industryis bagasse, which contains mainly cellulose, hemicellulose andlignin. Due to the high level of fibers, this material must be treat-ment before microbial utilization, which is routinely done byhydrolyzation. The possibility of bagasse utilization for PHAs syn-thesis was demonstrated by Yu and Stahl (2008). Employed in thiswork Ralstonia eutropha used hydrolyzed bagasse to PHAs synthe-sis, and accumulated it up to 57% of CDW.

Crude glycerol, themain by-product of biodiesel production, canbe used by bacteria as an inexpensive carbon source for the pro-duction of biodegradable polyhydroxyalkanoates (Mothes and Otto,


Table 1Fatty acid compositions of plant oils.

Vegetable oil Fatty acid (wt %) Reference

8:0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:0 Others

Cottonseed 28.7 0 0.9 13.0 57.4 0 0 0 Dermibas (1998)Peanut 11.4 0 2.4 48.3 32.0 0.9 1.3 3.7 Goering et al. (1982)Rapeseed 3.5 0 0.9 64.1 22.3 8.2 0 1.0 Isigigur et al. (1995)Soybean 12.0 3.0 23.0 55.0 6.0 0 1.0 Singh and Singh (2010)Sunflower 6.4 0.1 2.9 17.7 72.9 0 0 0 Dermibas (1998)Coconut 5.8 5.1 44.7 19.1 10.0 3.6 8.8 2.7 0.2 Bunyakiat et al. (2006)Palm oil 43.0 0.2 4.3 39.5 10.8 0.3 1.9 Melero et al. (2010)Palm kernel 3.8 3.6 48.4 17.4 9.1 2.7 12.6 2.4 0 Bunyakiat et al. (2006)Olive 0.01 12.1 1.2 3.0 72.8 9.5 1.39 Monfreda et al. (2012)Corn 12.0 2.0 25.0 6.0 55.0 Singh and Singh (2010)


2009). Promising results have been published by Cavalheiro et al.(2009), in which C. necator DSM 545 was cultured up to 68.8 gCDW/L on waste glycerol. PHB concentration reached 50% of celldry weight, with a productivity of 1.1 g/L/h. when the nitrogencontent was limited. Crude glycerol was also used by Hermann-Kraus et al. (2013), as the substrate in PHB-HV copolymer pro-duction by Haloferax mediterranei. In this work, a higher PHA con-centration was obtained with crude glycerol (16.2 g/L) than pureglycerol (13.4 g/L). Whereas Kawata and Aiba (2010) reported thatHalomonas sp. KM-1 utilized pure glycerol more effectively thanwaste form in PHB production, even though the initial glycerolcontent of the 2% pure glycerol culture was almost the same as thatof the 3% waste glycerol culture.

Recently, it was revealed that oilseed meal as by-product fromthe biodiesel industry could be successfully utilised for the pro-duction of PHAs. Studies applying fed-batch fermentations ofC. necator DSM 545 using rapeseed meal hydrolyzates as nutrientsupplement have demonstrated that the bacteria was capable tosynthesize up to 55.6% of P(3HB-co-3HV) (García et al., 2013). Thesame results have been reported by Kachrimanidou et al. (2013)with the same strain when the fermentation medium was sup-plemented with hydrolyzates from sunflower meal.

Various members of the grass family have been tested for bio-plastic production. Koutinas et al. (2007) and Xu et al. (2010) haveexamined the potential of a wheat-based biorefinery for the pro-duction of PHA. In the first report, Koutinas and co-workers showedthat hydrolyzed wheat mixed with fungal extract could produce upto 51.1 g/L PHB. Xu et al. (2010), reported that Cupriavidus necatorgrowing on wheat-derived media produced 162.8 g/L PHB. Grassbiomass has also been tested as a substrate formcl-PHA production.Cerrone et al. (2014) have found that P. putida contains 39% of CDWwhen cultivated on volatile fatty acids derived from aerobicallydigested ensiled grass. These PHAs were composed mainly of 3-hydroxydecanoic acid.

Recently, Jerusalem artichoke (Helianthus tuberosus) has beenproposed as a feedstock for PHB production. Koutinas et al. (2013)hydrolyzed Jerusalem artichoke tubers to fructose, glucose, aminoacids and peptides. This hydrolyzate was then tested as a substratefor PHB production in shake flask fermentation, resulting in anintracellular biopolymer content of up to 4 g/L. According to theauthors, it is possible to use hydrolyzed Jerusalem artichoke, butthe fermentation medium must be enriched with nutrient-richsupplements.

6. Plant oils as renewable resources

Plant oils such as soybean oil, palm oil and corn oil are desirablecarbon sources for PHAs production as they are relatively cheaperthan most sugars. It was reported that the theoretical yield of PHA


production from fatty acids is 0.65 g/g, whereas the theoreticalyield of PHA production from glucose ranges between 0.30 and0.40 g/g (Chanprateep, 2010).

The chemical composition of vegetable oils includes completeesters of glycerin and higher monocarboxylic acids (Table 1),therefore theymake excellence substrates for bioplastic productionby microbial fermentation. The demand for vegetable oils hasincreased rapidly in the past decade, mainly as the result of higherconsumption of edible oils and the development of the biodieselindustry. The global production of vegetable oils is still increasing(United States Department of Agriculture, 2013). Moreover, in thenear future “tailor-made” triacylglycerols, which can be subsitutesof lipids, could be produced on the base of waste fats by “non-conventional biocatalysis” or by oleaginous microorganisms (forreviews see: Papanikolaou and Aggelis, 2010, 2011; Metzger andBornscheuer, 2006).

Although plant oils can be used directly in the human diet and tofeed animals, they can also be used for a multiplicity of non-ediblepurposes. These substrates are used to efficiently produce variousadded-valuemetabolites such as organic acids or enzymes, which isof great interest to the industrial microbiology and biotechnologysectors. Organic acids have many industrial applications in the foodand pharmaceutical industry, such as additives for flavor, acidifi-cation and preservation. Among organic acids, citric acid is has thehighest industrial demand. Its synthesis, using Yarrowia lipolyticawith plant oil as a substrate, has been studied by Kamzolova et al.(2005). In their work, a yield of 135 g of citric acid per liter wasobtained when rapeseed oil was used. Lipases are the mostimportant class of industrial enzymes. They can be used to producedetergents, cosmetics, pharmaceuticals, flavor enhancers and foods(Cancino et al., 2008). The successful production of lipases usingolivemill wastewater and Y. lipolytica has been described byMoftahet al. (2013).

Recently, however, plant oil stocks have mostly been channeledinto biodiesel production (Adamczak et al., 2009). Biodiesel is apossible alternative to conventional petrol-based diesel and can beproduced by the transesterification of triacylglycerols (TAGs) fromvegetable oil and animal fats. Depending on the climate and soilconditions, different kinds of edible vegetable oils are being used asthe main feedstocks for biodiesel production, such as sunflower oilin Europe, soybean oil in the USA, rapeseed oil in Canada and palmoil in Southeast Asia (Bankovi�c-Ili�c et al., 2012). Although thecommercial use of biodiesel is still limited by its high price, we canexpect that global demand for vegetable oils will increase.

7. PHAs production from oils

Various plant oils have been tested and proven as effectivecarbon sources towards polyhydroxyalkanoates biosynthesis with



interesting material properties. The physical and material proper-ties of PHAs depend on their monomer composition and chemicalstructure for example the length of the pendent groups in thepolymer backbone, their chemical nature or the distance betweenthe ester linkages (Rai et al., 2011) (Fig. 3). It is well known that thestructure of the substrate, the PHAs system synthesis or thedegradation pathway of the carbon source have an influence onPHAs composition synthesized from fatty acids (de Ward et al.,1993). The type of monomer incorporated in the polymer chain isaffected by the microorganism used, the culture conditions and thetype of plant oil. There are several reports in the literature of PHAsproduction from oils. Table 2 summaries the types of PHAs andtheir contents produced by bacteria grown on plant oils.

7.1. Poly(3-hydroxybutyrate) homopolymer

P(3HB) is the most common polyhydroxyalkanoates in nature. Itwas shown that Cupriavidus necator H16 successfully accumulatedup to 79, 81 and 79% of CDW of P(3HB) on olive oil, corn oil andpalm kernel oil, respectively (Fukui and Doi, 1998). Other researchgroup demonstrated that Burkholderia sp. USM (JCM15050) wasable to synthesize 70% of P(3HB) when grown on crude palm kerneloil (Chee et al., 2010a). Also, other P(3HB) producers have beenidentified, however with the lowest biopolymers' biosynthesis.Kimura et al. (1999) reported polyhydroxybutyrate production atlevel of 49.8% of CDW when Chromobacterium sp. was culturedusing sunflower oil. Majid et al. (1999) employed Erwinia sp. USMI-20 for the production of P(3HB) using crude palm oil that resultedin the P(3HB) concentration at the level of 46% of CDW. Bhubalan

Fig. 3. Pure and waste oil as sub


et al. (2010) reported that Chromobacterium sp. USM 2 convertedpalm kernel oil into biopolymer with a much lower efficiencyachieving only 23% of CDW of P(3HB).

It has been also proven that the application of waste oils couldenhance P(3HB) homopolymer biosynthesis. However, there areonly few reports on P(3HB) production from waste oils. Taniguchiet al. (2003) demonstrated that Cupriavidus necator has the abilityto accumulate a high amount of P(3HB) (4.6 g/L) when waste ses-ame oil was employed as a sole carbon source. The same species hasbeen further investigated by Verlinden et al. (2011) who reportedthat waste oils could be a good alternative to purified oil orexpensive sugars for P(3HB) production. The authors have shownthat the achieved homopolymer concentration (1.2 g/L) fromwasterapeseed oil was similar to the concentration that has been ob-tained from glucose.

7.2. Copolymers

Besides P(3HB) homopolymer, biosynthesis of copolymers hadbeen also conducted using plant oils. The copolymers containing3HB as a constituent along with other HAs units of chain lengthsranging from 3 to 14 carbon atoms possess better properties andprocessability than P(3HB) homopolymer itself. Therefore, theirbiosynthesis has attracted attention of many researchers. Theincorporation of different monomer units into the poly-hydroxybutyrate chain generally requires expensive precursorswhich dose should be carefully controlled during cultivation due totheir common toxicity. The usage of inexpensive carbon sourcessuch as oils could decrease the overall biopolymers costs.

strates for PHAs production.


Table 2PHAs biosynthesis from renewable resources.

Strain PHA Feedstock Biomass(g/l)

PHA(%)

Fermentationmode

References

Recombinant Escherichia coli P(3HB) Whey 96.1 72.9 Fed-batch Nikel et al. (2006)Cupriavidus necator DSM 545 P(3HB) Whey permeate 8.5 18.0 Batch Povolo et al. (2009)Thermus thermophilus HB8 P(3HV-co-3HA) Whey 1.6 35.6 Batch Pantazaki et al. (2009Haloferax mediterranei P(3HB) Extruded rice bran and corn starch 140.0 55.6 Batch Huang et al. (2006)Bacillus megaterium P(3HB) Hydrolyzed cassava starch 4.97 29.7 Batch Krueger et al. (2012)Ralstonia eutropha NCIMB 11599 P(3HB) Waste potato starch 179.0 52.5 Fed-batch Haas et al., 2008Bacillus megaterium P(3HB) Cane molasses 1.3 40.0 Batch Gouda et al. (2001)Klebsiella aerogenes 2688 P(3HB) Sugarcane molasses 3.8 23.0 Fed batch Zhang et al. (1994)Recombinant Escherichia coli P(3HB) Sugarcane molasses 4.0 75.7 Fed-batch Saranya and Shenbagarathai (2011)Ralstonia eutropha P(3HB) Bagasse hydrolyzate 6.0 65 Batch Yu and Stahl (2008)Aspergillus awamori P(3HB) Crude Jerusalem Artichoke hydrolyzate 7.7 51.9 Batch Koutinas et al. (2013)Cupriavidus necator JMP 134 P(3HB) Crude glycerol 50 48 Fed-batch Mothes et al. (2007)Cupriavidus necator DSM 545 P(3HB) Crude glycerol 68.8 38 Fed-batch Cavalheiro et al. (2009)Cupriavidus necator P(3HB) Hydrolyzed wheat 29.9 58 Fed-batch Koutinas et al. (2007)Cupriavidus necator P(3HB) Wheat based biorafinery 15.8 44 Fed-batch Xu et al. (2010)Pseudomonas putida mcl-PHA Volatile fatty acids derived from

digested ensiled grass1.56 39.0 Batch Cerrone et al. (2014)


The member of PHA family, (3HB-co-3HV) copolymer, is one ofthe best characterized because of its high commercial potential.Some microorganisms were capable to biosynthesize P(3HB-co-3HV) copolymers from various crude plant oils, however it wasdependent on the type of 3HV precursors used. Lee and co-investigators (2008) demonstrated the production of P(3HB-co-3HV) copolymer by Cupriavidus necator H16 with 3HV molar frac-tion ranging from 3 to 14 mol% by adding sodium valerate andsodium propionate as the incorporators of 3HVmonomers. When amixture of palm kernel oil and sodium propionate was used thecopolymer content (90% of CDW) and biomass value (7.5 g/L) werethe highest with 3HV fraction of only 7 mol%. Whereas a combi-nation of olive oil and sodium valerate resulted in the highest 3HVmonomer composition (14 mol%) with a copolymer content of 89%of CDW. Also, waste rapeseed oil was found to be an excellentsubstrate for the growth and P(3HB-co-3HV) production byCupriavidus necator H16. Obruca et al. (2010) enhanced copolymerproduction by adding precursors such as propanol, propionate andvalerate. Propanol improved both biopolymer and biomass for-mation, up to 80% of CDW and 14.7 g/L of biomass was obtained.The highest 3HV molar fraction of 18 mol% was obtained throughthe feeding of valeric acid.

Among the vast number of different PHAs, P(3HB-co-4HB)copolymer is one of the most desirable biopolyester due to itsuseful mechanical properties that makes it applicable in the med-ical and pharmaceutical fields (Willimas and Martin, 2002). Basedon the literature, bacteria are able to incorporate 4-hydroxybutyrate monomer to form P(3HB-co-4HB) copolymergrown on plant oils. For example, Ralstonia eutropha KCTC 2662 hasbeen shown by Park and Kim (2011) to be able to produce up to 83%CDWof P(3HB-co-4HB) with 4HBmolar fraction in the range from 6to 10 mol% by adding soybean oil. This type of copolymer has beenalso successfully extracted by Rao and coworkers (2010) whenCuprividus necator was fed with spent palm oil along with 1,4-butanediol. The mentioned study revealed that the mole fractionof 4HB in the copolyester was constant at 15 mol% and did notdepend on the fermentation time.

The incorporation of 3-hydroxyhexanoate (3HHx) as the secondmonomer improved the properties of copolymer which are almostsimilar to those of the common plastics. P(3HB-co-3HHx) is aninteresting copolymer because its backbone consists of 3HB (an SCLmonomer) and 3HHx (an MCL monomer). Incorporation of smallamounts of 3HHx units (5 mol%) into the 3HB sequence leads tobetter thermal processability and physical properties of copolymer


(Matsusaki et al., 2000). A recombinant strain of Ralstonia eutrophaH16 harboring Aeromonas caviae PHA synthase gene, phaCAc, wasshown to produce a copolymer of 3HB with 5 mol% of 3HHx(P(3HB-co-5 mol% 3HHx), from soybean oil as a sole carbon sourcewith a high PHA content of 71e74% of CDW (Kahar et al., 2004). Thehighest 3HHx monomer fraction in the copolymer was reached byWong and coworkers (2012) who have evaluated different types ofplant oils in the cultivation of an engineered strain of Cupriavidusnecator Re2160/pCB113 harboring PHA synthase gene from Ral-stonia aetherivorans. The P(3HB-co-3HHx) copolymer productionon crude palm kernel oil was found to be themost suitable and gaveunexpectedly high 3HHx content of 68 mol%. The above mentionedachievements showed that copolymers could be produced from asingle substrate that is an inexpensive bio-based carbon sourceavailable in large quantities. It was also reported that the ability tothe microbes to convert waste plant oils into P(3HB-co-3HHx)copolymer is remarkable. In the recent study Kamilah et al.(2013) demonstrated that the transformant of Cupriavidus necatorharboring A. caviae PHA synthase gene was found to synthesize upto 85% of CDW of P(3HB-co-3HHx) copolymer grown on wastecooking oil resulting in the biomass concentration at 22.3 g/L.

The bacteria was found not only to be able to utilize plant oilsbut also was capable of producing a random terpolymer. A re-combinant strain of Cupriavidus necator harboring A. caviae PHAsynthase gene, was shown to accumulate 79% of P(3HB-co-3HV-co-3HHx) terpolymer grown on crude palm kernel oil. The 3HV molarfraction, ranged from 2 to 60 mol%, was controlled by the feedingtime and concentrations of sodium valerate or sodium propionateas the precursors (Bhubalan et al., 2008). In the separate study,Tsuge et al. (2009) have also observed the successful production ofanother terpolymer (3HB-co-3HO-co-3HD) by Ralstonia eutrophaharboring the wild-type Pseudomonas sp. 61-3 PHA synthase 1grown on soybean oil. The extracted PHA consisted of 91 mol% 3HB,5 mol% 3HO and 4 mol% 3HD.

7.3. Mcl-PHAs

Medium-chain-length polyhydroxyalkanoates have been suc-cessfully produced by many microorganisms grown on crude andwaste plant oils. Pseudomonads are undoubtedly the most versatileproducers of them. The first study reported mcl-PHAs productionfrom triacyloglycerols was carried out in the early 1990s. Cromwicket al. (1996) demonstrated the first usage of tallow as the solecarbon source for mcl-PHAs synthesis using Pseudomonas



resinovorans. This process was not profitable owing to the lowcontent of the produced PHAs, only 15% of CDW. Later, the moreefficiency biosynthesis processes were shown by using varioustypes of oils. Generally the ability to use plant oils towards mcl-PHAs production in associated to lipase activity. However, someauthors reported that bacteria able to produce lipase were unableto convert some plant oils into polyhydroxyalkanoates. In thesecases the additional saponification step must have been conducted(Silva-Queiroz et al., 2009; Mo _zejko and Ciesielski, 2013). Further-more, it was confirmed that the composition and level of mono-mers fraction of mcl-PHAs reflects the composition of plant oilused. It is well known that Pseudomonas species growing on fattyacids containing from 13-carbon to 18- carbon accumulate mono-mers consisting of 8-carbon and 10-carbon monomers (Madisonand Huisman, 1999). This tendency has been confirmed byseveral data regarding the composition of PHAs produced fromdifferent plant oils as the sole carbon sources.

Hazer et al. (1998) demonstrated the ability of P. oleovorans toaccumulate mcl-PHAs grown on oils such as hazelnut, sesame,olive. The obtained poly-3-hydroxyalkanoates contained repeatingunits from 8 to 20 carbon atoms. The highest mcl-PHAs yield (60%of CDW) was reported when the bacteria was cultivated on hazel-nut oil. In a separate study, Pseudomonas guezennei biovar tikehauhas been found to be able to utilize canola oil for the polymerproduction with a maximum mcl-PHAs yield of 63% of CDW(Simon-Colin et al., 2008). The researchers have also focused theirattention on the optimization of the biopolymer biosynthesis pro-cess using palm oil as a carbon source. The highest mcl-PHAscontent was revealed by Annuar et al. (2007) who achieved up to70% of PHAs cultivating P. putida PGA1 on saponified palm kernel oilin the fed-batch bioreactor. The authors reported that 3-hydroxyoctanoate (C8) and 3-hydroxydeconoate (C10) were themajor components of the extracted mcl-PHAs.

The production of mcl-PHAs fromwaste plant oils has also beenreported in the literature. Mo _zejko and coworkers (2011) foundthat the newly isolated strain Gl01, which belongs to Pseudomonasspecies was capable of producing 21% of mcl-PHAs on waste rape-seed oil. The similar PHAs concentration has been reached by Songet al. (2008) during a cultivation of Pseudomonas sp. DR2 usingwaste vegetable oils. In these cases, the monomeric composition ofpolyesters was found to consist mainly of 3-HO and 3-HD. Inter-estingly, Pseudomonas sp. DR2 grown on fried oil was not able tosynthesized 3HDD monomer although a small amount of 3HHxD(5.24 mol%) was detected. Based on the literature, waste palm oilseemed to be also a suitable substrate for mcl-PHAs biosynthesis.Investigations applying fed-batch fermentations of Pseudomonassp. Gl01 using saponified waste palm oil have shown that the strainwas able to synthesize up to 48% of mcl-PHAs at 17 h of the culture.The produced mcl-PHAs contained monomers such as 3-HHx, 3-HDD, and 3-HTD. Furthermore, the authors reported a high molarfraction of 3HN (up to 63 mol%) what indicated the presence ofuneven fatty acids in the waste palm oil used (Mo _zejko andCiesielski, 2013). Similarly, when waste frying oil was supplied asa substrate to Pseudomonas aeruginosa 42A2 by Fern�andez et al.(2005), a trace amount of 3-HN (0.47%) was detected in theextracted mcl-PHAs, however the biopolymer content was lower(29.4% of CDW).

Besides Pseudomonas species, also Comamonas testosteroni hasbeen tested for its ability to produce mcl-PHA from oils such ascastor oil, coconut oil, cottonseed oil, mustard oil, olive oil, sesameoil. This PHA producer was able to accumulate biopolymers up to87.5% of CDW with major monomer compositions consisting of3HO and 3HD (Thakor et al., 2005). The productivity of biopolymersreferring to the conversion rate of oil tomcl-PHAswas high reachedthe values above 50%.


8. Material properties of polyhydroxyalkanoates from oils

The biopolymer properties determine which applications theseplastics are best suited for and how they can be processed. ThePHAs applicability depends on their thermal, physical and me-chanical properties such as melting temperature, glass transitiontemperature, molecular weights, tensile strength or Young's mod-ule (Fig. 4). These properties can be triggered by fine-tuning of thecomposition of the PHAs during the biosynthesis.

Scl-PHAs, such as poly-3-hydroxybutyrate (PHB), show a highcrystallinity and strong brittleness with poor elastic properties,which restrict their use as a stable and useful material. The smalldifference between the decomposition temperature (approxi-mately 270 �C) and the high melting point (around 180 �C)restricted its processability for melt extrusion technology. Me-chanical properties such as the Young's modulus and the tensilestrength are close to those of polypropylene. However, theextension to break for P(3HB) is much lower than that of syn-thetic plastics. When copolymer formation occurs with 3HV, 4HBor 3HHx monomer units, the properties of the material alterresult in better mechanical features, a decrease in stiffness or anincrease in toughness. Mcl-PHAs accumulated by many Pseudo-monas species have more favorable properties than scl-PHAs.They are elastomers with a low degree of crystallinity with alow melting temperature, a low tensile strength and a highelongation to break. Moreover, PHAs possess various propertieswhich make them interesting to industry such as: non-toxic,biocompatible and insoluble in water. Table 3 summarizes mo-lecular weights and thermal properties of various PHAs producedby bacteria grown on plant oils.

The molecular weight of polyhydroxyalkanoates is believed tobe the most important factor affecting their properties. Its value isdependent on the bacterial host, the medium composition and thesubstrate used or the fermentation conditions. Lee et al. (2008)have found crude palm kernel oil is a suitable substrate for theproduction of high molecular weight of P(3HB-co-3HV) copolymer.A decrease of 3HV molar fraction from 10 to 8 increased thenumber-average molecular weight (Mn) from 7.0 � 105 to9.9 � 105 g/mol, respectively. Whereas the value of Mn and Mw/Mnof P(3HB-co-3HHx) copolymer accumulated by Cupriavidus necatorH16 grown on soybean oil were 4.3 � 105 g/mol and 2.5, respec-tively (Thakor et al., 2005). The incorporation of 3-hydroxyvalerateimproves mechanical properties of biopolymers. It was demon-strated that the yield of only 10% 3-HV influences significantly onelongation to break from 3% [P(3HB) homopolymer] to 20% (P(3HB-co-3HV) copolymer.

The incorporation of 4HB monomers results in copolymershaving better elastomeric properties than homopolymer P(3HB). Itwas confirmed that P(3HB-co-4HB) copolymer extracted from thecells of Cupriavidus necator grown on spent palm oil showed amelting peak at 160.8 �C, glass transition temperature at �2.4 �Cand crystallization temperature at 46.9 �C (Rao et al., 2010). Addi-tional analysis employed to evaluate the biocompatibility of P(3HB-co-4HB) blends revealed that the copolymer have the potential tobe developed as a new absorbable biomaterial for medicalapplications.

Wong et al. (2012) reported that the highest 3HHx monomerfraction is the lowest molecular weight P(3HB-co-3HHx) copol-ymer produced by recombinant Cupriavidus necator fed with palmoil. The mechanical properties of P(3HB-co-3HV) copolymer aredependent on themolar ratio of 3HV. The value of Young's Modulusdecreases with an increase of 3HV from 0 to 25 mol%, thus indi-cating that P(3HB-co-3HV) becomes more flexible. Furthermore,the tensile strength decreases gradually as the 3HV molar ratioincreases. Further, the tensile strength and Young's modulus of the


Fig. 4. The properties of PHAs depend on their structures.


P(3HB-co-3HHx) blends decreased from 7.91 to 0.13 Mpa and100.96 to 0.27 Mpa, respectively, as the HHxmonomer fractionwasincreased from 32 to 70 mol%. The above mentioned valuesrevealed that the copolymers tested are soft and flexible. The highcontent of 3HHx have greatly increased the elasticity of thecopolymer and resulted in the higher elongation to break(1074.60%) than low-density polyethylene (LDPE) (700%) (Doi,1990).

Number and weight average molecular weights of mcl-PHAsfrom oils are relatively lower compared to those of scl-PHAs.Haba et al. (2007) reported that P. aeruginosa 47T2 grown onwaste cooking oil was able to produce mcl-PHA with the Mn andMw value ranged from 34.3 to 36.7 kDa and from 37.1 to 38.8 kDa,respectively. These results were consistent with the data ofMo _zejko and Ciesielski (2013), who cultured Pseudomonas sp. Gl01using saponified waste palm oil and Hazer et al. (1998) who syn-thesized mcl-PHAs from P. oleovorans grown on hazelnut oil. Ashbyand Solaiman (2008) demonstrated that supplementation of crudehydrolyzed oil led to the higher molecular weights values of mcl-PHAs from P. resinovorans NRRL B-2649 (Mn ¼ 156 kDa). Howev-er, the low molecular weight of biopolymers could be used as acomponent of polymeric emulsifiers or to form block copolymers.

Glass and melting transition temperatures are important pa-rameters relative to in-service applications of PHAs. They definelower and upper temperature limits for numerous applications,


especially for semicrystalline polymers. Canola oil was found to be agood substrate for the production of P(3HB) homopolymer byCupriavidus necator (L�opez-Cellular et al., 2010). DSC thermogramsshowed a PHB melting temperature (Tm) of 170 �C with DHm of84.74 J/g, these data are in accordance with others in the literature(Furukawa et al., 2005; Loo et al., 2005). The analysis of P(3HB-co-3HV) obtained from the different plant oils showed the presence ofthe melting peak in the range of 166e177 �C. The lowest meltingtemperature was found when the culture was supplemented witholive oil and this peak corresponded to the copolymer containinghigh 3HV monomer. The glass transition temperature decreasedfrom �1 to �12 �C as the 3HHx monomer concentration in theP(3HB-co-3HHx) copolymer increased from 32 to 70mol%. Tg valueof mcl-PHAs ranged from-61.7 �C to �14 �C and decreased with anincrease in the length of the monomers side chain. van der Walleet al. (2001) reported a decrease of Tg value of approximately18 �C for mcl-PHAs biosynthesized from coconut fatty acids(�43.7 �C) to that produced from linseed oil fatty acid(Tg ¼ �61.7 �C). The highest Tm of the mcl-PHAs from waste oilsreported so far was 86 �C (Mo _zejko and Ciesielski, 2014). Thesevalue was lower compared to those biopolymers produced byPseudomonas sp. Gl01 grown on waste rapeseed oil (Mo _zejko et al.,2012), P. guezennei grown on coprah oil (Simon-Colin et al., 2008) orP. aeruginosa 42A2 cultured on waste frying oil (Fern�andez et al.,2005).


Table 3PHAs synthesized by microorganisms grown in oily carbon sources.

PHA Bacteria fermentation mode Oil Biomass(g/l)

PHAcontent(%)

PHA yield(g/l)

Mw x104

(g/mol)Mn x104

(g/mol)Tm(�C)

Tg(�C)

References

Crude plant oilsP(3HB) Cupriavidus necator

H16 Fed batchSoybean oil 126.0 76.0 95.7 125.4 33.0 nd nd Kahar et al. (2004)

P(3HB-co-3HV) Cupriavidus necator Batch Palm oil 3.6 74.0 2.7 168.0 67.0 170 �0.9 Lee et al. (2008)P(3HB-co-3HV) Pseudomonas oleovorans Batch J.curcas seed oil nd 26.1 nd 16.7 13.2 160 179 Allen et al. (2010)P(3HB-co-70% 3HHx) Cupriavidus necator Batch Coconut oil 2.61 48.0 1.3 22.7 13.7 no �12 Wong et al. (2012)P(3HB-co-3HHx-co-3HO) Cupriavidus necator mutant Batch Soybean oil nd 57.0 nd 18.7 8.5 126 �4 Tsuge et al. (2009)mcl-PHA Pseudomonas putida PGA1 Batch Palm kernel oil 3.0 37.0 1.1 12.4 5.88 no �45 Tan et al. (1997)mcl-PHA Pseudomonas oleovorans

ATCC 29347 BatchHazelnut oil 2.0 60.0 1.2 9.1 5.9 nd nd Hazer et al. (1998)Sesame oil 2.9 41.0 1.2 11.0 6.8 nd ndOlive oil 1.2 26.0 0.31 6.7 4.5 nd nd

mcl-PHA Pseudomonas resinovoransBatch

Olive 3.4 43.1 1.5 11.9 7.2 nd nd Ashby andFoglia (1998)Sunflower 3.1 41.2 1.3 11.2 6.5 nd nd

Coconut 3.8 51.0 1.9 16.5 10.1 nd ndSoybean 2.9 44.5 1.3 12.7 7.0 nd nd

mcl-PHA Pseudomonas chlororaphisHS21 Batch

Palm kernel oil 3.3 45.0 1.4 12.7 8.3 no �44 Sun et al. (2003)

mcl-PHA Pseudomonas aeruginosa Batch Linseed oil 3.6 50.2 1.8 12.6 6.0 no �51 Bassas et al. (2008)mcl-PHAs Pseudomonas guezennei

biovar.tikehauTwo step fed-batch

Coprah oil nd 63.0 nd nd nd 45 �45 Simon-Colinet al. (2008)

mcl-PHA Pseudomonas aeruginosa Batch B. carinata oil 1.0 5.0 0.05 5.6 3.1 no �47 Impallomeniet al. (2011)

Waste plant oilsP(3HB-co-3HV) Cupriavidus necator H16 Batch Waste cooking oil 4.3 75.0 3.2 136.0 34.0 171 �0.1 Lee et al. (2008)P(3HB-co-4HB) Cupriavidus necator Batch Waste palm oil 5.5 81.0 4.4 nd nd 160.8 �2.4 Rao et al. (2010)mcl-PHAs Pseudomonas aeruginosa

47T2 BatchWaste cooking oil 21.0 36.0 7.6 3.88 2.42 no �60.5 Haba et al. (2007)

mcl-PHAs Pseudomonas sp. Gl01Fed-batch

Waste palm oil 3.8 43.0 1.6 10.9 6.5 86 �55 Mo _zejko andCiesielski (2013)

mcl-PHAs Pseudomonas sp. Gl01Fed-batch

Waste rapeseed oil 4.4 44.0 2.0 14.4 5.8 46 �38 Mo _zejko andCiesielski (2014)

nd e not determined; no e not observed.


9. Concluding remarks and future prospects

PHAs are useful and valuable materials that can be used inmany industrial fields due to their versatile properties. However,these biopolymers are not widely used because of their high price,which arises from the high costs of the substrates used for theirproduction. Therefore, in recent years many studies have focusedon using low-cost agricultural wastes as fermentative carbonsources, which could improve the economics of this process.Agricultural feedstocks are also attractive because they arerenewable resources, which makes PHAs production environ-mentally friendly. Although vegetable oils in both pure and spentforms seem to be attractive carbon sources for PHA production,little has been reported on the costs of producing them from oilysubstrates. One source reports that the costs of carbon sourcesconstitute 40% of the final cost of biopolymers produced by bac-terial fermentation (Obruca, 2010). If pure oil is used, the price ofPHAs will be higher than that of synthetic plastic. However, usingwaste substrates reduces the total financial outlay, which meansthat the final cost of PHAs could be competitive with the price ofpolypropylene. As previously reported, the price of rapeseed isaround 3.51 V/kg (Obruca et al., 2010), whereas its waste equiva-lent is costs about half of that (Mo _zejko et al., 2011). Also, theentire integrated process of PHAs production should be consideredbecause other factors such as up-stream and down-stream pro-cesses can influence the final price of PHAs. 30% of the overallproduction costs come from the recovery of PHAs from bacterialcells (Sun et al., 2007). Some separation methods are especiallyexpensive, such as solvent extraction, which involves extra costsfor disposal of used solvent.


There are a number of advantages to the use of plant oils forPHAs production. Global production of these oils is still increasing,which could make themmajor sources of agricultural feedstocks inthe future. Because these oil crops can be grown in any warm ortemperate region of the world, this process would be geopoliticallyindependent. In addition, using waste oils for PHA production helpsreduce the costs and problems associated with disposal of theseoils. Bioconversion of wastes like these into valuable products likepolyhydroxyalkanoates is one of the most attractive aspects of theemerging global bio-economy. Using waste oils to produce PHAscan diversify incomes in the agricultural sector by allowing it todeliver plant oils to the biotechnology market. In order to realizethese advantages, a number of things must be determined, such asthe proper proportions of ingredients, storage issues, and the reli-ability of rawmaterials. An important factor could be the amount ofavailable waste oils. In addition, transporting these oils over longdistances might be problematic and costly.

The process of PHAs production from plant oils can be mademore efficient by utilizing all parts of oilseed plants. Currently,mainly the seeds are used, whereas the rest of the plant is dis-carded. Harvesting the plants produces large amounts of wastebiomass, and then processing the plants for oil production usuallycreates oil cake or oil meal as waste by-products. These wastescould be used as substrates for anaerobic digestion for the pro-duction of biogas. The biogas could be used to heat the bioreactorswhere the PHAs are produced. This integration of PHAs productionwith biogas production is an attractive integrated technologyconcept (Fig. 5).

There is not much information available about using wasteplant oils to produce PHAs because most studies have used pure


Fig. 5. Integrated technology concept combining plant oils conversion into PHAs and biogas production from residues such as plant biomass, oil cakes and oil meals.


fatty acids. Furthermore, there is a lack of data about the prop-erties of PHAs produced with waste oils. To predict the physicaland processing characteristics of these PHAs, it is necessary toknow at least their molecular weight, melting temperature andglass transition temperature. Detailed information about thePHAs molecular weight distribution is needed to provide infor-mation about the polymer and allow predictions of otherimportant characteristics like tensile strength, melt viscosity, andmodulus of elasticity. Even if the extracted PHAs have the sametensile strength or melt viscosity, their mechanical propertieswill differ due to variations in molecular weight distributions.Finally, little is known about how feeding strategies influence theyield of PHA extraction. In most cases, plant oils have been testedas potential carbon sources in shake flask experiments. Morereliable results would be obtained by fermentation in bioreactorswhere important parameters like dissolved oxygen, temperatureand pH can be monitored throughout the process. The datarevealed in experiments like these could be used in planningindustrial processes.

Information on the efficiency of PHA synthesis with oily sub-strates is ambiguous, which can be discouraging for potential in-vestors interested in their use. Contradictions arise from differentmethods used for evaluating the process efficiency of PHAs pro-duction. In most cases, PHAs content in bacterial cells has beenevaluated with gas chromatography results, although some re-searchers have weighed PHAs after extraction and purification.Furthermore, reports of biopolymer productivity have differed evenwhen using the same type of oil because different bacterial strainswere used. There is a need for more studies that evaluate theproductivity of individual strains that are grown in culture mediasupplemented with different carbon sources. These studies willprovide information about what kind of oil is most suitable forbiopolymer production. In addition, more studies are needed withwaste oils from frying. When these oils are exposed to high tem-peratures, food and air for long periods, chemical reactions canform compounds toxic to bacteria. Thus, the chemical


characteristics and fatty acid composition of frying oils should bedetermined to optimize the PHAs synthesis process.

Investigations are also needed to better understand and over-come technical difficulties in the use of oils for producing PHAs.One major drawback is foam formation, which decreases the con-centration of dissolved oxygen. This is overcome by adding anti-foam reagents, but it is not known how these reagents reduce orincrease bacteria growth rates.

Oils also create problems with downstream processes to obtainpurified PHAs, which means that in addition to the need fordeveloping cell breakage procedures, more investigations areneeded on how to separate PHAs from cultures with oily substrates.Thus, there is a need to develop and optimize new fermentationstrategies and novel methods of product recovery.

In summary, to introduce PHAs to the global market, theremust first be improvement in fermentation processes with cheapfatty carbon sources, biopolymers purification technologies, andthe development of recombinant bacteria to lower the price ofPHAs so that it will be close not only to other biomaterials likepolylactide but also to the conventional plastics that are anessential part of almost all industries. Second, the drawbacks inthe properties of PHAs need to be overcome. The range of theirproperties could be extended by producing composites andblends. This has the potential to make PHAs suitable for manyapplications. Several facts speak for PHA-based materials. Theseare the potential to innovate new economic opportunities, tooffset limited fossil fuels supplies and to help solve the problem ofwaste management. Do PHAs have the chance to be an importantcommodity to enhance the comfort and quality of our life? TheUnited States Food and Drug Administration (FDA) has recentlyapproved P(4HB) for the clinical applications, which is a huge stepforward and suggests a promising future for these biopolymers.PHAs are likely to meet the requirements of specific market ap-plications and become the biomaterials of the 21st century. Tomake this a reality, the potential of bacterial species and recom-binant strains that can convert plant oils into valuable PHAs



should be further studied in the context of PHAs yield, theirproperties and the ease of their purification.

This review summarizes the most recent advances in poly-hydroxyalkanoates production using plant oils. This paper consti-tutes the unique source of information on the possible usage ofdiverse oily substrates towards PHAs production. Furthermore, arelationship between a type of oil and PHAs properties has beenshown. Current knowledge presented in this paper will be usefulfor both entrepreneurs who are interested in the utilization of oilsand scientists who look for ideal replacement of petrochemicalbased plastic.

Acknowledgments

The authors thank Mark Leonard for help with English grammarand usage. This work was financially supported by European Unionunder project nr UDA-POKL.04.01.01-00-095/10-00.

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Plant oils as promising substrates for polyhydroxyalkanoates production

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