International Journal of Molecular Sciences Review Carbon Sources for Polyhydroxyalkanoates and an Integrated Biorefinery Guozhan Jiang 1, David J. Hill 1, Marek Kowalczuk 1,2, Brian Johnston 1, Grazyna Adamus 2, Victor Irorere 1 and Iza Radecka 1,* 1 School of Biology Chemistry and Forensic Science, University of Wolverhampton, Wolverhampton WV1 1LY, UK; [email protected] (G.J.); [email protected] (D.J.H.); [email protected] (M.K.); [email protected] (B.J.); [email protected] (V.I.) 2 Polish Academy of Sciences, Centre of Polymer and Carbon Materials, Zabrze 41-819, Poland; [email protected] * Correspondence: [email protected]; Tel.: +44-1902-322-366 Academic Editor: Carl Joseph Schaschke Received: 19 May 2016; Accepted: 11 July 2016; Published: 19 July 2016 Abstract: Polyhydroxyalkanoates (PHAs) are a group of bioplastics that have a wide range of applications. Extensive progress has been made in our understanding of PHAs’ biosynthesis, and currently, it is possible to engineer bacterial strains to produce PHAs with desired properties. The substrates for the fermentative production of PHAs are primarily derived from food-based carbon sources, raising concerns over the sustainability of their production in terms of their impact on food prices. This paper gives an overview of the current carbon sources used for PHA production and the methods used to transform these sources into fermentable forms. This allows us to identify the opportunities and restraints linked to future sustainable PHA production. Hemicellulose hydrolysates and crude glycerol are identified as two promising carbon sources for a sustainable production of PHAs. Hemicellulose hydrolysates and crude glycerol can be produced on a large scale during various second generation biofuels’ production. An integration of PHA production within a modern biorefinery is therefore proposed to produce biofuels and bioplastics simultaneously. This will create the potential to offset the production cost of biofuels and reduce the overall production cost of PHAs. Keywords: polyhydroxyalkanoates; microorganisms; carbon sources; biorefinery 1. Introduction Polyhydroxyalkanoates (PHAs) are a group of bacterial polyesters produced by a variety of prokaryotic microorganisms under unbalanced nutrition conditions as carbon and energy storage materials [1]. PHAs, due to their superior biodegradability, biocompatibility and thermal processability [2,3], are currently produced by many companies, such as Metabolix® (Woburn, MA, USA), Procter & Gamble Co., Ltd. (Cincinnati, OH, USA), Tianjin Green Bioscience Co., Ltd. (Tianjin, China) and Biocycle PHB Industrial SA (Serrano, SP, Brazil) [2]. PHA production capacity has increased rapidly over the last decade, which according to statistics from the European Bioplastics Association, reached up to 34,000 tonnes of global production capacity in 2013 [4]. The U.K. share of this potential market is currently £2 million [5]. However, the expanding PHA industry has raised concerns, since most PHAs are produced primarily from food crops, sugar cane and vegetable oils, as listed in Table1. Production of these carbon sources competes with food supply production. For example, when corn is used as the carbon source for substrate glucose, one kilogram of corn can produce 0.67 kg of glucose [6], and this amount of glucose can produce 0.27 kg of PHAs [7]. Consequently, 34,000 tonnes of PHAs would utilise Int. J. Mol. Sci. 2016, 17, 1157; doi:10.3390/ijms17071157 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 1157 2 of 21 Int. J. Mol. Sci. 2016, 17, 1157 2 of 20 about126,000 126,000 tonnes tonnes of corn. of corn.Therefore, Therefore, it is itessentia is essentiall to toexploit exploit non-food-based non-food-based carbon carbon sources sources for for a sustainable production. Table 1. Main polyhydroxyalkanoate (PHA)(PHA) producers andand theirtheir substrate.substrate. MainMain Product Product ProductionProduction Capacity Capacity CompanyCompany MainMain Substrate Substrate (Brand)(Brand) (Tons/Year)(tons/year) Biomer (Krailling, Germany) Sucrose [8] P3HB Not available Biomer (Krailling, Germany) Sucrose [8] P3HB Not available Bio-on (Bologna, Italy) Beet sugar [9] MINERV®-PHA 10,000 Bio-on (Bologna, Italy) Beet sugar [9] MINERV®-PHA 10,000 TianjinTianjin Green Green Bio Bio (Tianjin, (Tianjin, China) China) SucroseSucrose [ 8[8]] P3HBP3HB 10,00010,000 KanekaKaneka (Takasago, (Takasago, Japan) Japan) VegetableVegetable oil oil [ 8[8]] PHBHPHBH 10001000 BiocycleBiocycle PHA PHA Industrial Industrial (Serrano, (Serrano, Brazil) Brazil) CaneCane sugar sugar [ 10[10]] P3HBP3HB 100100 Tian’anTian’an (Ningbo, (Ningbo, China) China) CornCorn [ 8[8]] P3HBP3HB 10,00010,000 MetabolixMetabolix (Woburn, (Woburn, MA, MA, USA) USA) CornCorn [ 11[11]] Mirel™-PHAMirel™-PHA 50,00050,000 PHAPHA = Polyhydroxyalkanoate, = Polyhydroxyalkanoate, P3HB P3HB = =Poly-3-hydroxybutyrate, Poly-3-hydroxybutyrate, PHBH PHBH = = poly-3-hydroxybutyrate-co- poly-3-hydroxybutyrate-co- 3-hydroxyhexanoate.3-hydroxyhexanoate. In this paper, we will review PHA production in terms of substrates substrates and the the derivation derivation processes processes of the substrates substrates from from various various carbon carbon sources. sources. The The aim aim of this of this study study is to is identify to identify opportunities opportunities and andconstraints constraints related related to the to thefuture future sustainable sustainable production production and and toto assess assess the the framework framework for for the implementation of the productionproduction withinwithin aa biorefinery.biorefinery. For convenience in later discussions,discussions, the structure of PHAsPHAs isis shownshown inin FigureFigure1 .1. TheseThese structurally-diversestructurally-diverse polymerspolymers areare composedcomposed ofof hydroxyalkanoic units with different numbers of carbon atoms by varying R and x.. In the following text, the abbreviations ofof thethe namesnames ofof PHAsPHAs areare used.used. HH O C OH * H C x C n R H O Figure 1.1. TheThegeneral general structure structure of of a polyhydroxyalkanoatea polyhydroxyalkanoate (PHA) (PHA) macromolecule. macromolecule. All monomeric All monomeric units haveunits have one chiral one chiral centre centre (*) with (*) with the the configuration configuration of of the theR Renantiomer, enantiomer, and and the the whole wholemacromolecule macromolecule has a perfectperfect isotactic structure [12]. [12]. The number of carbon atoms in the R substituentsubstituent ranges up to 18 [[13];13]; x rangesranges fromfrom 1–3,1–3, andand nn rangesranges fromfrom 100–30,000100–30,000 [[3,14].3,14]. ForFor poly(3-hydroxybutyrate)poly(3-hydroxybutyrate) (P3HB), (P3HB), R = CH 3 andand xx == 1; 1; for for poly(3-hydroxyvalerate) poly(3-hydroxyvalerate) (P3HV), (P3HV), RR == C C22HH5 5andand x x= =1;1; etc. etc. 2. Overview Overview of of Substrates Substrates for for Polyhydroxyalkanoates Polyhydroxyalkanoates (PHAs) (PHAs) Production Production PHAs are synthesized in bacterial cells through a metabolic process. The substrates for biosynthesizing PHAs PHAs are are usually usually restricted restricted to to small small molecules, molecules, since since bacteria bacteria have have thick, thick, rigid rigid cell cellwalls walls surrounding surrounding membranes. membranes. Large Large polymeric polymeric molecules molecules cannot cannot be transported be transported into the into cell, the and cell, andan extracellular an extracellular transformation transformation either either by the by micr the microorganismoorganism or by or a by chemical a chemical process process is needed is needed for forthe theuse use of ofthe the polymeric polymeric molecule molecules.s. A Asummary summary of of the the substrates substrates for for PHA PHA synthesis synthesis and typical microorganisms that can useuse thethe substratessubstrates isis givengiven inin FigureFigure2 2.. As shown shown in in Figure Figure 2,2 ,the the substrates substrates can can be gene be generallyrally divided divided into intothree three categories: categories: simple simple sugars sugars(monosaccharides), (monosaccharides), triacylglycerol triacylglycerol and hydrocar and hydrocarbons.bons. Most MostPHA-producing PHA-producing microorganisms microorganisms can canuse usesimple simple sugars, sugars, whilst whilst triacylglycerol triacylglycerol have have only only been been reported reported for for some some microorganisms. Hydrocarbon metabolism isis more limited,limited, butbut can be used by Pseudomonas species of bacteria. For the same substrate, differentdifferent bacteria can produce PHAsPHAs withwith aa differentdifferent composition.composition. For example, Pseudomonas spp.spp. can can use use glucose glucose and and other other simple simple sugars sugars to synthesize to synthesize medium medium chain-length chain-length PHAs, PHAs,such as such poly(3-hydroxylhexanoate) as poly(3-hydroxylhexanoate) (P3HHx) (P3HHx) [15–17], [15 while–17], whileRalstoniaRalstonia eutropha eutropha synthesizessynthesizes only onlypoly(3-hydroxybutyrate) poly(3-hydroxybutyrate) (P3HB) (P3HB) using using glucose glucose [18]. [18]. To better understand the conversion of various substrates to PHAs in Figure 2, one can refer to the catalytic conversion of substrates in a chemical process. In the biosynthesis of PHAs, the bacterial Int. J. Mol. Sci. 2016, 17, 1157 3 of 21 Int. J. Mol.Tobetter Sci. 2016 understand, 17, 1157 the conversion of various substrates to PHAs in Figure2,