Conversion of waste cooking oil into medium chain polyhydroxyalkanoates in a high cell density fermentation

Journal Pre-proof Conversion of waste cooking oil into medium chain polyhydroxyalkanoates in a high cell density fermentation Carolina Ruiz, Shane T Kenny, Tanja Narancic, Ramesh Babu, Kevin O’ Connor

PII:

S0168-1656(19)30838-7

DOI:

https://doi.org/10.1016/j.jbiotec.2019.08.020

Reference:

BIOTEC 8499

To appear in:

Journal of Biotechnology

Received Date:

28 May 2019

Revised Date:

20 August 2019

Accepted Date:

30 August 2019

Please cite this article as: Ruiz C, Kenny ST, Narancic T, Babu R, Connor KO, Conversion of waste cooking oil into medium chain polyhydroxyalkanoates in a high cell density fermentation, Journal of Biotechnology (2019), doi: https://doi.org/10.1016/j.jbiotec.2019.08.020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Conversion of waste cooking oil into medium chain polyhydroxyalkanoates in a high cell density fermentation

Carolina Ruiz1, Shane T Kenny2, Tanja Narancic1,4, Ramesh Babu3 and Kevin O’ Connor1,4,*

1

UCD Earth Institute and School of Biomolecular and Biomedical Science, University College Dublin,

Belfield, Dublin 4, Ireland. 2

Bioplastech Ltd., Nova UCD, Belfield Innovation Park, University College Dublin, Belfield, Dublin 4,

ro of

Ireland 3

AMBER Centre, CRANN Institute, School of Physics, Trinity College Dublin, Dublin 2, Ireland

4

-p

BEACON - Bioeconomy Research Centre, University College Dublin, Belfield, Dublin 4, Ireland`

Corresponding author

re

*Professor Kevin E. O’Connor, School of Biomolecular and Biomedical Sciences and BEACON -

lP

Bioeconomy Research Centre, O’Brien Centre for Science, University College Dublin, Belfield, Dublin

Highlights

ur

Valorisation of waste cooking oil through conversion into polyhydroxyalkanoate PHA. A high cell density bioprocess with high PHA volumetric productivity was developed. PHA composition reflects the fatty acid composition of the waste cooking oil used.

Jo

  

na

4, Ireland; Telephone: +353 1 716 2198, Fax: +353 1 716 1183, E-mail: [email protected]

Abstract

Biodegradable and biocompatible polymers polyhydroxyalkanoates (PHAs) have a wide range of applications from packaging to medical. For the production of PHA at scale it is necessary to develop a high productivity bioprocess based on the use of a cheap substrate. The objective of the current study was to develop a high cell density bioreactor-based process for the production of medium chain length polyhydroxyalkanoate (mclPHA) with waste cooking oil as the sole carbon and energy

1

source. A number of substrate feeding strategies for bacterial growth and polymer production were investigated. Pseudomonas chlororaphis 555 achieved high biomass of 73 g/L medium and a good biomass yield (including PHA in the cell) of 0.52 g/g substrate. P. chlororaphis 555 accumulated 13.9 g mclPHA/L and achieved polymer productivity of 0.29 g mclPHA/(L h). The mclPHA contained predominantly (R)-3-hydroxyoctanoic acid and (R)-3-hydroxydecanoic acid monomers, with a high fraction of (R)-3-hydroxydodecanoic acid monomers. This polymer is of low molecular weight (18 324

ro of

kDa), low polydispersity, it is amorphous, and has a glass transition temperature of -64C.

Keywords: Biocatalysis, Pseudomonas, biodegradable polymer, polyhydroxyalkanoate, waste cooking oil, high cell density, bioprocess development.

-p

Keywords: Biocatalysis, high cell density fermentation, bioreactor, Pseudomonas,

re

polyhydroxyalkanoate, waste cooking oil

lP

1. Introduction

Naturally occurring polymers like polyhydroxyalkanoates (PHAs) have a wide range of applications

na

due to their biodegradable, elastomeric, thermoplastic and biocompatible features (Nair and Laurencin 2007). Given these properties, PHAs have become a possible alternative material to

ur

synthetic, non-degradable plastic materials. PHAs can be produced from renewable resources such as sugars (Albuquerque et al. 2007), cellulose, starch (Nawrath et al. 1995), plant oils (Fukui and Doi

Jo

1998) and some waste products (Kenny et al. 2008; Pittmann and Steinmetz 2016; Revelles et al. 2017; Verlinden et al. 2011; Ward et al. 2006). However, the production cost of PHA is relatively high compared to conventional petrochemical plastics. This cost is driven mainly by carbon substrate cost and polymer productivity (Choi and Lee 1997; Sabapathy et al. 2017). Since PHA is an intracellular polymer, its productivity is also dependent on biomass productivity. Waste cooking oils (WCO) are non-edible due to chemical changes in the oil during the frying process, and so must leave the food chain. 29 million tonnes are produced per year, or about 4.1 kg

2

per person per year worldwide (Chuah et al. 2016; Maddikeri et al. 2012). Disposal of WCO is a major problem, due to poor end-of-life management, which can lead to pollution of water and land resources (Nantha Gopal et al. 2014). The use of WCO for PHA production can contribute to a variety of environmental benefits, such as biodegradable material manufacture from waste resources and an end-of-life management option (Maddikeri et al. 2012; Tangy et al. 2017). At present, WCO is predominantly managed through incineration and while this provides energy, it is a suboptimal use of a valuable carbon. This resource could be better used to make materials, thus extend the residence time of carbon in the materials cycle and contribute to resource efficiency (Taniguchi et al.

ro of

2003). While biodiesel has been looked at as a route for WCO management (Pezzella et al. 2016; Wang et al. 2006), a critical and costly impediment to the use of WCOs as feedstocks for biodiesel production is the presence of high free fatty acids (FFA). FFAs significantly reduce the quality of the

-p

biodiesel (Akaraonye et al. 2010; Aslan A. N. et al. 2016; Riedel et al. 2015). Many researchers are therefore looking for alternative uses for WCOs, from detergents, to coatings and lubricants

re

(Chowdhury et al. 2013; Pezzella et al. 2016; Shimizu K. 1988; Shogren et al. 2004; Smith H. et al.

lP

2013).

WCO has been identified as a substrate for PHA accumulation (Fernández et al. 2005; Haba et al.

na

2007; Kourmentza et al. 2018; Song et al. 2008; Verlinden et al. 2011). Pseudomonas aeruginosa has been shown to produce medium chain length (mcl) PHA from WCO (Fernández et al. 2005a; Vidal-

ur

Mas et al. 2001; Haba et al. 2007). However, viewed as an opportunistic pathogen, P. aeruginosa could pose several challenges for PHA production at an industrial scale. Critically, high cell density

Jo

cultivation of PHA accumulating microorganisms, a prerequisite for an industrial process, has not been reported with WCOs. In a previous study we reported that P. chlororaphis 555 grows and produces PHA from pure plant oils (Walsh et al. 2015). The aim of this study was to investigate bioprocess strategies in stirred tank bioreactors to achieve high cell density, high mclPHA accumulation and high mclPHA volumetric productivity by P. chlororaphis 555 using WCO as the substrate. P. chlororaphis strains in general

3

lack known toxic properties, and have a history of safe use in agriculture. Finally, the properties of the polymer produced under the best bioprocess conditions were examined.

2. Materials & Methods 2.1 Bacterial strain and culture conditions Pseudomonas chlororaphis 555 was maintained on Pseudomonas isolation agar (PIA, Sigma-Aldrich, Ireland). Liquid minimal salt media (MSM; per 1 l of diH2O: 9 g Na2HPO4x12H2O, 1.5 g KH2PO4 and 3.9

ro of

g NH4(SO4)2 was used for growing pre-cultures and bioreactor experiments. After autoclaving, 1 ml of trace element solution (per 1 l of 1 M HCl: 4 g ZnSO4x7H2O, 10g FeSO4x7H2O, 1 g CuCl2 x 2H2O, 1 g MnCl2x4H2O, 1 g Na2B4O7x10H2O, 0.2 g NiCl2x6H2O, 0.3 g Na2MoO4x2H2O, 2 g CaCl2, 1.2 g

-p

(NH4)5Fe(C6H4O7)2) and 1 ml of a 1 M MgSO4x7H2O were aseptically added to the medium. For the cultivation under phosphorous limitation 6.4 g Na2HPO4x12H2O and 1.1 g KH2PO4 were used.

re

2.2 Bioreactor conditions

lP

A bioprocess using P. chlororaphis 555 was assessed using both batch and pulse-fed batch strategies with WCO as the carbon and energy source. A single colony from a PIA plate was inoculated into 50

na

ml MSM supplemented with 3 g WCO/l. This pre-inoculum was incubated at 30°C, with shaking at 200 rpm for 16-18 hours. The pre-inoculum (0.5 ml) was then seeded into fresh 50 ml MSM supplemented with 3 g WCO/l and cultivated for 16-18 hours under the same conditions as described

ur

above. 200 ml (4 x 50 ml) of this culture was used as the inoculum for a 5 l stirred tank bioreactor

Jo

(Sartorius Biostat B+, Germany) containing 3 l MSM. The initial agitation was set at 500 rpm and stirring was controlled up to a maximum of 1500 rpm to maintain dissolved oxygen (DO) at or above 20%. pH was controlled at 6.9 +/- 0.1 by addition of 20% NH4OH solution or 15% (v/v) H2SO4. The NH4OH also acted as a nitrogen source. Foaming was controlled by addition of antifoam solution (polypropylene glycol P2000, Sigma). WCO was supplied as the sole carbon substrate at specified concentrations of 60, 90 and 120 g WCO/l for batch bioreactor experiments and a total of between

4

140 g WCO/l and 220 g WCO/l were supplied with 20 g pulses of WCO per litre for fed batch bioreactor experiments. 2.3 Determination of free fatty acids in waste cooking oil (WCO) All WCO used in this study was supplied by Frylite® (Orchard Road Industrial Estate, Strabane, Northern Ireland). This oil was collected as waste from various catering establishments in Ireland. To quantify the fatty acids in WCO, 2 g of WCO were dissolved in 40 ml of a 50:50 (vol:vol) mixture of diethylether and ethanol. 2 drops of 1% phenolphthalein indicator (in ethanol) were added and the

A=

CNaOHVNaOHMW OA msample1000

ro of

mixture was titrated against 0.01 M NaOH.

100 (wt %)

-p

A: Acid value, CNaOH: concentration of sodium hydroxide in moles per litre, VNaOH: volume of

re

sodium hydroxide in milliliters used, MWOA: molecular weight oleic acid, m: mass in grams of oil sample

lP

2.4 Analysis of fatty acids composition of waste cooking oil

Fatty acids from chemically hydrolysed waste cooking oil were derivatised with N-Methyl-N-

na

(trimethylsilyl)trifluoroacetamide (TMS). 2 ml of chloroform was placed in a gas chromatography vial. 1 µl of the substrate and 20 µl of TMS were added. The vial was placed at 70°C for 30 minutes. The

ur

fatty acids were then analysed using an Agilent 6890N gas chromatograph (GC) fitted with a 5973

Jo

series inert mass spectrophotometer (MS). A HP-5 column (12 m x 0.2 mm x 0.33 µm; Hewlett Packard) was used with an oven method of 50°C for 3 min, increasing by 10°C /min to 250°C and holding for 1 min. A 10:12 split was used with helium as the carrier gas and an inlet temperature of 250 ˚C. 2.5 Nutrient and biomass analysis Two 2 ml samples were taken at 1 or 2 h time intervals from each growth and PHA accumulation experiment. The samples were centrifuged at 16000 x g for 3 min. The supernatant was retained for

5

phosphate concentration analysis and the cell pellet was then frozen at -80°C, lyophilised (Labconco, Fisher Scientific) until dry and weighed. Soluble phosphate concentration and the concentration of nitrogen in the supernatant were determined using previously described protocols (EPA 1978; Scheiner 1976). 2.6 PHA content and monomer composition determination from bacterial cultures PHA content of lyophilised cell material was determined by acidic methanolysis as previously described (Lageveen et al. 1988). In brief, 5-10 mg of dried cells were resuspended in 2 ml of acidified

ro of

methanol (15% H2SO4, v/v) and 2 ml of chloroform containing 6 mg/l benzoate methyl ester as an internal standard, and incubated at 100C for 3 hours. The 3-hydroxyalkanoic acid methyl esters were extracted by water and assayed by gas chromatography (GC) using a Hewlett-Packard 6890 N chromatograph equipped with a HP-Innowax capillary column (30m×0.25mm, 0.50μm film thickness;

-p

Agilent Technologies) and a flame ionisation detector (FID). A temperature program was used to

re

separate the different 3-hydroxyalkanoic acid methyl esters (120°C for 5 min, increasing by 3°C/min to 180°C, 180C for 10 min. A 20:1 split was used with helium as the carrier gas and an inlet

lP

temperature of 250C. For the peak identification, commercially available 3-hydroxyalkanoic acids (Bioplastech Ltd, Dublin, Ireland) were methylated as described above for PHA samples. Total PHA

Jo

ur

analysis.

na

content was determined as a percentage of cell dry weight (CDW) using the peak areas from GC

2.7 Polymer isolation Cells were harvested from cultivations in a Sorvall centrifuge (Fisher Scientific, Dublin, Ireland) at 25,000 x g, frozen at −80°C for 24 h and lyophilised. Room temperature acetone (100 ml) was mixed with 10 g of cells for 24 h. The mixture was allowed to settle, and the supernatant was filtered using a 0.2 μm PTFE filter. Acetone was then evaporated under vacuum (Buchi, Switzerland) until approximately 90 ml of acetone had been recovered. The polymer was precipitated using 2 vol of a

6

wash solution consisting of 35% methanol, 35% ethanol and 30% distilled water (Elbahloul and Steinbuhel 2009). The supernatant was then decanted, and the precipitated PHA was allowed to dry before further analysis. 2.8 Gel permeation chromatography (GPC) and differential scanning calorimetry (DSC) of PHA polymers The average molecular weight (Mw), the molecular number (Mn) and the polydispersity index (PD) of the polymer were measured by GPC (Viscotek 305 TDA, Malvern UK) using Porous styrene

ro of

divinylbenzene copolymer gel 8 mm analytical columns with triple detector array (TDA). Spectroscopic grade tretahydrofuran was used as the eluent with a flow rate of 1.0 ml/min. Sample concentration of 1% (w/v) and injection volumes of 100 µl were used. A molecular weight calibration curve was generated with two polystyrene standards of molecular weight 108 KDa and 245 KDa

-p

having low polydispersity (Kenny et al. 2008).

re

The polymer was analysed by DSC with a Perkin-Elmer Pyris Diamond calorimeter calibrated to Indium standards to determine the glass transition temperature (Tg), melting temperature (Tm), and

lP

degradation temperature (Td). The samples were encapsulated in hermetically sealed aluminium pans and heated from -80 to 100°C at a rate of 10°C/min. To determine the glass transition

na

temperature (Tg) the samples were held at 100°C for 1 min and rapidly quenched to -70°C. The samples were then reheated at 10°C/min starting from -80°C and ending at 100°C to determine the

ur

melting temperature (Tm) and Tg. Finally, the samples were heated to 350°C at a rate of 10°C/min to

Jo

determine the Td (Kenny et al. 2008). 3. Results

3.1 Properties of waste cooking oil (WCO) The WCO used in this study is a mixture of oils collected from restaurants in Ireland. In WCO unsaturated fatty acids predominate but saturated fatty acids make up over 24% of total fatty acid content (Table 1, entry 1).

7

In addition to measuring the glycerol bound fatty acid content of the WCO, the free fatty acid (FFA) content, present due to hydrolysis of oils during cooking was also analysed. FFA can act as carbon and energy source for bacteria and so can contribute to biomass and PHA accumulation. FFA composition of WCO was very similar to the glycerol bound fatty acid composition (data not shown). However, based on titration with 0.01M NaOH we found that the FFA content of WCO was very low (0.4% w/w). 3.2 Batch bioreactor experiments with WCO

ro of

An assessment of WCO utilisation by P. chlororaphis 555 in a batch bioreactor was undertaken using 60, 90 and 120 gWCO /l (Table 2). The culture reached approximately 45 gCDW/l and 19% of CDW as PHA when 60 gWCO/l was used. When 90 gWCO/l was supplied both biomass and PHA showed a 1.2and 1.4-fold increase in biomass and PHA (Table 2). Finally, when the concentration was further

-p

increased to 120 gWCO/l the biomass remained the same as when 90 gWCO/l was supplied, while a

re

1.2-fold decrease in PHA was observed (Table 2).

In all these bioreactor experiments no limitation of either nitrogen or phosphorus was observed, and

lP

oxygen was still available in excess, which indicates that some other factor was inhibiting growth when higher levels of WCO were supplied. While it would have been valuable to determine how

na

much WCO was remaining at the end of these experiments, it was not possible to accurately measure the concentration of WCO due to the heterogeneous physical dispersion of the oil in the

ur

liquid medium and a propensity for the oil to stick to sampling ports devices and tubes. The gravimetric method to measure used cooking oil (UCO) depletion in a bioreactor used by Cruz and

Jo

colleagues showed deviations of up to 50% (Cruz et al. 2015b). Several methods of oil extraction followed by gravimetric analysis or analysis of fatty acids by GC were attempted, but all resulted in an error greater than 50%. In the absence of these data, biomass and PHA yields were calculated based on the total oil added (Table 2). The best yield for biomass was achieved when 60 gWCO/l was supplied in a 30-h fermentation. While experiment using 90 gWCO/l had lower biomass yields, it showed a similar PHA accumulation yield when compared to the 60 gWCO/l experiment (Table 2).

8

The PHA accumulated by P. chlororaphis 555 from WCO contained (R)-3-hydroxydodecanoic acid (C12), (R)-3-hydroxydecanoic acid (C10), (R)-3-hydroxyoctanoic acid (C8), (R)-3-hydroxyhexanoic acid (C6) monomers in a ratio of 20:37:36:7. 2-fold more (R)-3-hydroxydodecanoic acid is present in PHA accumulated by cells supplied with WCO compared to cells grown with oleic acid (ratio of C12:C10:C8:C6 = 12:39:41:8). Oleic acid is a reference substrate as it is present in triglycerides of WCO and it is known to be a substrate for PHA accumulation by bacteria. 3.3 Pulse fed batch fermentation of P. chlororaphis 555 supplied with WCO

ro of

Pulse fed-batch fermentations were then developed based on growth data from P. chlororaphis 555 in batch fermentations. While our results from batch experiments showed that the strain can

tolerate 60 and 90 g WCO/l, in the fed batch bioreactor pulses of WCO greater than 20 g/l negatively affected the performance of the dissolved oxygen probe. As the feeding of WCO to the bioreactor

-p

was based on the rise in dissolved oxygen (DO-stat) we limited the pulse of WCO to 20 g/l.

re

The substrate was fed when the DO2 began to rise above the set point of 20% DO2, indicating the bacteria were running out of substrate. In total, 220 g WCO was supplied per litre of fermentation

lP

medium over the 48 hours. The growth medium was designed to avoid any inorganic nutrient limitation and had an initial phosphorus concentration of 1.1 gP/l. The maximum biomass of 73

na

gCDW/l was achieved at time 44 h but this decreased to 62.1 gCDW/l by 48 h potentially due to cell lysis (Fig. 1A). This equates to the biomass yield of 0.28 g/g (Table 4). As no nutrient limitation was

ur

imposed in this fermentation, only a low level of PHA was observed (Fig. 1A) giving a PHA yield of

Jo

0.03 g/g (Table 3).

In feed strategy 2 (FS2) the same amount of WCO was supplied to the bioreactor as in the FS1, however phosphorous limitation was imposed to stimulate PHA accumulation (Fig. 1B). The phosphorous concentration was reduced to 0.8 gP/l. Using FS2 a 1.3-fold increase in the final total biomass (residual cells and PHA combined) and 1.9-fold increase in PHA was achieved (Fig. 1B). This resulted in a total biomass yield 1.3-fold higher and PHA yield 2.6-fold higher compared to FS1 (Table

9

3). FS2 also resulted in the highest PHA volumetric productivity of 0.36 g/l/h (Table 3). Complete phosphorous depletion occurred by 33 hours in FS2 (data not shown). As phosphorous limitation improved PHA accumulation, the subsequent feed strategies (FS3 and FS4) were designed to improve the bioprocess yields by combining phosphorus limitation and lower amounts of WCO supplied to avoid WCO over feeding. Over the lifetime of the fermentation using FS3 200 g WCO was supplied in total per litre of fermentation medium, while FS4 supplied a total of 140 g WCO/l over the 48 h fermentation. FS3 achieved similar biomass to FS1, however the PHA level was 1.5-fold lower (Fig. 1C). While the final biomass achieved in FS4 was 1.1-fold lower when

ro of

compared with FS1 (Fig. 1D), this was the most successful feeding strategy with regard to biomass and PHA yield (Table 3). The overall biomass and PHA yield were 1.9- and 3.6-fold improved

compared to FS1 (Table 3). The residual WCO at the end of the bioprocess based on FS4 was 7  5

-p

g/l. In all feeding strategies PHA accumulation peaked around 20 hours and then fluctuated but did

re

not significantly increase above the value observed at time 20 h (Fig. 1A-D). 3.4 Characterisation of PHA accumulated by P. chlororaphis 555 when grown on WCO

lP

The PHA isolated from P. chlororaphis 555 grown using WCO as the sole carbon and energy source is completely amorphous, as evidenced by the lack of a defined Tm under the conditions tested (Table

na

4). The polymer is very sticky at room temperature and has a very low glass transition temperature (60°C). Furthermore, the polymer has a low molecular mass and a polydispersity (Mw/Mn) of 1.9

ur

(Table 4).

Jo

4. Discussion

WCO was chosen for this study as a cheap substrate because the price of the carbon source is a major contributor to the cost of PHA production (Fernández et al. 2005; Muhr et al. 2013). Compared to all previous reports, Pseudomonas chlororaphis 555 used in the current study has achieved by far the highest biomass and mclPHA productivity when using waste cooking oil (WCO). The dissolved oxygen (DO-stat) strategy was chosen due to the highly hydrophobic nature of the substrate and consequent inaccuracy in substrate utilisation analysis. Therefore, dissolved oxygen was used to

10

indirectly monitor the substrate consumption i.e. the demand for oxygen decreases when the substrate is consumed. In this study, the substrate feeding occurred when dissolved oxygen increased above 20%. DO-stat based feeding strategies have been successfully employed for high cell density fermentations and at scale processes (Jing et al. 2018; Lee et al. 2000). 3.8- to 7.2-fold higher biomass was achieved in this study compared to the biomass achieved by Pseudomonas Gl01 fed with glucose and waste rapeseed oil in a two-step fed-batch fermentation (Mozejko et al. 2012). While a high content of PHA (66.1% of CDW) was achieved when Pseudomonas aeruginosa NCIB 40045 was grown aerobically in a shake flask with waste free fatty acids from

ro of

soybean oil (Fernández et al. 2005), the cell dry weight was not reported. To the best of our

knowledge, this is the first time that high cell density growth in a bioreactor using WCO was

achieved. Furthermore, the PHA volumetric productivities achieved using FS2 and FS4 (Table 4) are

-p

2- to 2.5-fold higher than that achieved with P. chlororaphis DSM50083 (0.14 g/l/h) grown on tallow-

re

based biodiesel (fatty acid methylesters) (Muhr et al. 2013). Martino and colleagues reported that Cuprivadus necator cultivated with WCO reached 10.4 g/l biomass and accumulated 37% PHB in a

lP

batch fermentation in 27 hours (Martino et al. 2014). While the sclPHA content was 1.9-fold higher compared to approximately 20% CDW PHA achieved by P. chororaphis 555 in this study (Table 3), the

na

final biomass of C. necator was 4.3-fold lower resulting in a 1.3-fold lower PHA volumetric productivity (0.14 g/l/h) compared to PHA volumetric productivity achieved in the current study

ur

(Table 3). Other reports of growth and PHA accumulation using Pseudomonas species include flask experiments with P. resinovorans NRRL B-2649 where 3.2 g/l biomass and 0.9 g/l PHA were achieved

Jo

(Cruz et al. 2016b), P. aeruginosa 47T2 cultivated in a submerged culture with WCO where 7.6 g/l of PHA was observed (Haba et al. 2007) which is 2.3-fold lower than the PHA production achieved with FS2 in the current study. A two-step fermentation of P. resivorans with a mixture of waste apricots and/or grapes and WCO resulted 10.2 g/l biomass with 12.4% of PHA (Follonier et al. 2014). Lipases and esterases have role in bacterial hydrolysis of triacylglycerols, the main component of WCO, into glycerol and fatty acids, which are then metabolised by central metabolic pathways (Rosenau and Jaeger 2000). The lipases from Pseudomonas species have been extensively

11

characterised and applied in biotechnological processes (Rios et al. 2018). P. chlororaphis 555 is likely equipped with highly active lipase(s), allowing it to efficiently utilise WCO. However, the production of lipases by this strain and their activity in WCO hydrolysis remains to be determined. While the batch fermentation with 60 gWCO/l gave the highest biomass yield (0.76 g/g; Table 3), the final residual biomass and PHA was much lower compared to fed batch feeding strategies. Interestingly, the PHA content of cells (% of CDW) in fed batch strategies was not higher than the batch fermentations (Table 3 and 4). One hypothesis for the inability to increase productivities is that the use of the low value waste by-products such as WCO in bioreactors drastically changes the

ro of

rheology of the fermentation with an increase in viscosity and shear-thinning behaviour with loss of homogeneity. This in turn is making it difficult to keep control of the standard parameters and thus affects intracellular PHA accumulation due to problems of mass transfer once high cell densities are

-p

achieved (Freitas et al. 2013).

re

The complexity of WCO can affect the composition of PHA and thus the properties of the PHA polymer (Gui et al. 2008; Vastano et al. 2015). We used WCO from a commercial collector (Frylite®)

lP

of WCO from restaurants throughout the island of Ireland and is thus a good regional representative of WCO. WCO used in this study had a higher content of unsaturated fatty acids (Table 1) which is

na

broadly in keeping with other studies (Cruz et al. 2015a; Leung and Guo 2006; Martino et al. 2014). However, saturated fatty acids were present at higher levels than some previous reports (Table 1)

ur

(Abidin et al. 2013; Leung and Guo 2006; Martino et al. 2014). Palm oil and animal fat have higher levels of saturated fatty acids (Knothe et al. 2015; Mba et al. 2015), and therefore their use could

Jo

have contributed to the higher presence of saturated fatty acids in the WCO used in this study . The fatty acid content of the substrate is important as this will affect the physical characteristics of the waste cooking oil which affects bioavailability in an aqueous liquid medium, but it also affects the monomer composition of the PHA polymer accumulated by the bacteria (Gillis et al. 2017). The monomer composition of the PHA accumulated by P. chlororaphis 555 from WCO was consistent throughout the cultivation period and contained a much higher (R)-3-hydroxydodecanoic acid

12

compared to cells grown with oleic acid. This could be due to the presence of fatty acids such as stearic acid in WCO that may affect bacterial metabolism. Few studies attempting to convert WCO to PHA have examined the properties of the produced polymer. PHA extracted from P. resinovorans grown with olive oil deodorizer distillate was also liquid at room temperature but had a significantly higher glass transition temperature (-16°C) which could be due to the higher molecular weight of that polymer (Cruz et al. 2016a) compared to the PHA produced by P. chlororaphis 555 in the current study (Table 4). Similarly, P. resinovorans grown with soybean oil produced PHA that was amorphous and showed no melting transition but again had a

ro of

higher glass transition temperature (-45°C) (Ashby and Foglia 1998). In addition, molecular weight and molecular number of PHA produced from WCO were lower in comparison with PHA produced using P. chlororaphis 555 and rapeseed oil (Table 4, entries 2 and 3). It is likely that the components

-p

present in WCO have some inhibitory effect on the PHA metabolism, however further investigation is

re

required to determine this. Relatively low molecular weight and molecular number also characterise PHA isolated from P. putida KT2440 cultivated with hydrolysed WCO (Table 4, entry 4) (Ruiz et al.

lP

2019).

na

5. Conclusions

We have demonstrated the capacity of P. chlororaphis 555 and pulse fed batch fermentation using

ur

WCO directly as the sole source of carbon and energy to achieve the highest reported biomass and

Jo

PHA content, PHA titre g/l and PHA volumetric productivity. The solubility of WCO in the aqueous medium may be limiting PHA accumulation in the later stages of the bioprocess where cell density is higher than at the early stages of growth. The future plan is to understand the exact enzymes involved in the hydrolysis of WCO by P. chlororaphis 555, which will allow us to engineer the strain and potentially improve the turnover of WCO into PHA. While the biomass achieved is high, engineering solutions such as bioreactor design could be investigated to increase WCO availability

13

which can contribute to even higher biomass and PHA productivity when P. chlororaphis 555 uses WCO as a substrate.

Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

ro of

Acknowledgements Carolina Ruiz was funded under the Brazil Ireland CAPES programme (ref 9147136). Tanja Narancic acknowledges support under European Union Horizon 2020 research and innovation programme

-p

633962 for the project P4SB. Ramesh Babu was funded by Science Foundation Ireland SFI Grant No.

Jo

ur

na

lP

re

SFI/12/RC/2278.

14

References

Jo

ur

na

lP

re

-p

ro of

Abidin, S.Z., Patel D., Saha B., 2013. Quantitative analysis of fatty acids composition in the used cooking oil (UCO) by gas chromatography-mass spectrometry (GC-MS). Can J Chem Eng 91:1896-1903 doi:10.1002/cjce.21848 Akaraonye, E., Keshavarz T., Roy I., 2010. Production of polyhydroxyalkanoates: the future green materials of choice. J Chem Technol Biot 85:732-743 doi:10.1002/jctb.2392 Albuquerque, M.G.E., Eiroa M., Torres C., Nunes B.R., Reis M.A.M., 2007. Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses. J Biotechnol 130:411-421 doi:10.1016/j.jbiotech.2007.05.011 Ashby, R.D., Foglia T.A., 1998. Poly(hydroxyalkanoate) biosynthesis from triglyceride substrates. Appl Microbiol Biot 49:431-437 doi:DOI 10.1007/s002530051194 Aslan A. N., Ali M. M., Morad N., P. T., 2016. Polyhydroxyalkanoates production from waste biomass. IOP Conference Series: Earth environmental sciences publishing 36:012040 Choi, J.I., Lee S.Y., 1997. Process analysis and economic evaluation for poly(3hydroxybutyrate) production by fermentation. Bioprocess Eng 17:335-342 doi:DOI 10.1007/s004490050394 Chowdhury, A., Mitra D., Biswas D., 2013. Biolubricant synthesis from waste cooking oil via enzymatic hydrolysis followed by chemical esterification. Journal of Chemical Technology & Biotechnology 88:139-144 doi:10.1002/jctb.3874 Chuah, L.F., Yusup S., Aziz A.R.A., Klemeš J.J., Bokhari A., Abdullah M.Z., 2016. Influence of fatty acids content in non-edible oil for biodiesel properties. Clean Technologies and Environmental Policy 18:473-482 doi:10.1007/s10098-015-1022-x Cruz, M.V., Araujo D., Alves V.D., Freitas F., Reis M.A.M., 2016a. Characterization of medium chain length polyhydroxyalkanoate produced from olive oil deodorizer distillate. Int J Biol Macromol 82:243-248 doi:10.1016/j.ijbiomac.2015.10.043 Cruz, M.V., Freitas F., Paiva A., Mano F., Dionisio M., Ramos A.M., Reis M.A.M., 2016b. Valorization of fatty acids-containing wastes and byproducts into short- and mediumchain length polyhydroxyalkanoates. New Biotechnol 33:206-215 doi:10.1016/j.nbt.2015.05.005 Cruz, M.V., Sarraguca M.C., Freitas F., Lopes J.A., Reis M.A.M., 2015a. Online monitoring of P(3HB) produced from used cooking oil with near-infrared spectroscopy. J Biotechnol 194:1-9 doi:10.1016/j.jbiotec.2014.11.022 Cruz, M.V., Sarraguça M.C., Freitas F., Lopes J.A., Reis M.A.M., 2015b. Online monitoring of P(3HB) produced from used cooking oil with near-infrared spectroscopy. Journal of Biotechnology 194:1-9 doi:https://doi.org/10.1016/j.jbiotec.2014.11.022 Elbahloul, Y., Steinbuhel A., 2009. Large-scale production of poly(3-hydroxyoctanoic acid) by Pseudomonas putida GPo1 and a simplified downstream process. Appl Environ Microb 75:643-651 doi:10.1128/Aem.01869-08 EPA (1978) Method 365.3: Phosphorous, all forms (colorimetric, ascorbic acid, two reagent). https://www.epa.gov/sites/production/files/2015-08/documents/method_3653_1978.pdf. 2017 Fernández, D. et al., 2005. Agro-industrial oily wastes as substrates for PHA production by the new strain Pseudomonas aeruginosa NCIB 40045: Effect of culture conditions. Biochem Eng J 26:159-167 doi:10.1016/j.bej.2005.04.022 Follonier, S., Goyder M.S., Silvestri A.C., Crelier S., Kalman F., Riesen R., Zinn M., 2014. Fruit pomace and waste frying oil as sustainable resources for the bioproduction of

15

Jo

ur

na

lP

re

-p

ro of

medium-chain-length polyhydroxyalkanoates. Int J Biol Macromol 71:42-52 doi:10.1016/j.ijbiomac.2014.05.061 Freitas, F., Alves V.D., Coelhoso I., Reis M.A. (2013) Production and food applications of microbial biopolymers. In: Teixeira J. A., A. VA (eds) Engineering aspects of food biotechnology. CRC Press, Boca Raton. doi:https://doi.org/10.1201/b15426 Fukui, T., Doi Y., 1998. Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain. Appl Microbiol Biot 49:333-336 doi:DOI 10.1007/s002530051178 Gillis, J., Ko K., Ramsay J.A., Ramsay B.A., 2017. Potential for mcl-PHA production from nonanoic and azelaic acids. Canadian journal of microbiology 64:11-19 Gui, M.M., Lee K.T., Bhatia S., 2008. Feasibility of edible oil vs. non-edible oil vs. waste edible oil as biodiesel feedstock. Energy 33:1646-1653 doi:10.1016/j.energy.2008.06.002 Haba, E., Vidal-Mas J., Bassas M., Espuny M.J., Llorens J., Manresa A., 2007. Poly 3(hydroxyalkanoates) produced from oily substrates by Pseudomonas aeruginosa 47T2 (NCBIM 40044): Effect of nutrients and incubation temperature on polymer composition. Biochem Eng J 35:99-106 doi:10.1016/j.bej.2006.11.021 Jing, K., Tang Y.W., Yao C.Y., del Rio-Chanona E.A., Ling X.P., Zhang D.D., 2018. Overproduction of L-tryptophan via simultaneous feed of glucose and anthranilic acid from recombinant Escherichia coli W3110: Kinetic modeling and process scale-up. Biotechnol Bioeng 115:371-381 doi:10.1002/bit.26398 Kenny, S.T. et al., 2008. Up-Cycling of PET (Polyethylene Terephthalate) to the Biodegradable Plastic PHA (Polyhydroxyalkanoate). Environ Sci Technol 42:7696-7701 doi:10.1021/es801010e Knothe, G., Krahl J., Van Gerpen J. (2015) The biodiesel handbook. Elsevier, Kourmentza, C., Costa J., Azevedo Z., Servin C., Grandfils C., De Freitas V., Reis M.A.M., 2018. Burkholderia thailandensis as a microbial cell factory for the bioconversion of used cooking oil to polyhydroxyalkanoates and rhamnolipids. Bioresource Technol 247:829-837 doi:10.1016/j.biortech.2017.09.138 Lageveen, R.G., Huisman G.W., Preusting H., Ketelaar P., Eggink G., Witholt B., 1988. Formation of polyesters by Pseudomonas oleovorans - Effect of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3hydroxyalkenoates. Appl Environ Microb 54:2924-2932 Lee, S.Y., Wong H.H., Choi J.I., Lee S.H., Lee S.C., Han C.S., 2000. Production of mediumchain-length polyhydroxyalkanoates by high-cell-density cultivation of Pseudomonas putida under phosphorus limitation. Biotechnol Bioeng 68:466-470 doi:Doi 10.1002/(Sici)1097-0290(20000520)68:4<466::Aid-Bit12>3.0.Co;2-T Leung, D.Y.C., Guo Y., 2006. Transesterification of neat and used frying oil: Optimization for biodiesel production. Fuel Process Technol 87:883-890 doi:10.1016/j.fuproc.2006.06.003 Maddikeri, G.L., Pandit A.B., Gogate P.R., 2012. Intensification approaches for biodiesel synthesis from waste cooking oil: A review. Ind Eng Chem Res 51:14610-14628 doi:10.1021/ie301675j Martino, L., Cruz M.V., Scoma A., Freitas F., Bertin L., Scandola M., Reis M.A.M., 2014. Recovery of amorphous polyhydroxybutyrate granules from Cupriavidus necator cells grown on used cooking oil. Int J Biol Macromol 71:117-123 doi:10.1016/j.ijbiomac.2014.04.016 Mba, O.I., Dumont M.-J., Ngadi M., 2015. Palm oil: processing, characterization and utilization in the food industry–a review. Food bioscience 10:26-41

16

Jo

ur

na

lP

re

-p

ro of

Mozejko, J., Wilke A., Przybylek G., Ciesielski S., 2012. Mcl-PHAs produced by Pseudomonas sp Gl01 using fed-batch cultivation with waste rapeseed oil as carbon source. J Microbiol Biotechn 22:371-377 doi:10.4014/jmb.1106.06039 Muhr, A. et al., 2013. Novel description of mcl-PHA biosynthesis by Pseudomonas chlororaphis from animal-derived waste. J Biotechnol 165:45-51 doi:10.1016/j.jbiotec.2013.02.003 Nair, L.S., Laurencin C.T., 2007. Biodegradable polymers as biomaterials. Prog Polym Sci 32:762-798 doi:10.1016/j.progpolymsci.2007.05.017 Nantha Gopal, K., Pal A., Sharma S., Samanchi C., Sathyanarayanan K., Elango T., 2014. Investigation of emissions and combustion characteristics of a CI engine fueled with waste cooking oil methyl ester and diesel blends. Alexandria Engineering Journal 53:281-287 doi:https://doi.org/10.1016/j.aej.2014.02.003 Nawrath, C., Poirier Y., Somerville C., 1995. Plant Polymers for Biodegradable Plastics Cellulose, Starch and Polyhydroxyalkanoates. Mol Breeding 1:105-122 doi:Doi 10.1007/Bf01249696 Pezzella, C., Vastano M., Casillo A., Corsaro M.M., Sannia G., 2016. Production of bioplastic from waste oils by recombinant Escherichia coli: a pit-stop in waste frying oil to biodiesel conversion race. Environ Eng Manag J 15:2003-2010 doi:DOI 10.30638/eemj.2016.216 Pittmann, T., Steinmetz H., 2016. Potential for polyhydroxyalkanoate production on German or European municipal waste water treatment plants. Bioresource Technol 214:9-15 doi:10.1016/j.biortech.2016.04.074 Revelles, O., Beneroso D., Menendez J.A., Arenillas A., Garcia J.L., Prieto M.A., 2017. Syngas obtained by microwave pyrolysis of household wastes as feedstock for polyhydroxyalkanoate production in Rhodospirillum rubrum. Microb Biotechnol 10:1412-1417 doi:10.1111/1751-7915.12411 Riedel, S.L., Jahns S., Koenig S., Bock M.C.E., Brigham C.J., Bader J., Stahl U., 2015. Polyhydroxyalkanoates production with Ralstonia eutropha from low quality waste animal fats. J Biotechnol 214:119-127 doi:10.1016/j.jbiotec.2015.09.002 Rios, N.S., Pinheiro B.B., Pinheiro M.P., Bezerra R.M., dos Santos J.C.S., Goncalves L.R.B., 2018. Biotechnological potential of lipases from Pseudomonas: Sources, properties and applications. Process Biochem 75:99-120 doi:10.1016/j.procbio.2018.09.003 Rosenau, F., Jaeger K.E., 2000. Bacterial lipases from Pseudomonas: Regulation of gene expression and mechanisms of secretion. Biochimie 82:1023-1032 doi:Doi 10.1016/S0300-9084(00)01182-2 Ruiz, C., Kenny S.T., Babu R., Walsh M., Narancic T., O’Connor K.E., 2019. High cell density conversion of hydrolysed waste cooking oil fatty acids into medium chain length polyhydroxyalkanoate using Pseudomonas putida KT2440. MDPI Catalysts 9:468-482 doi:doi:10.3390/catal9050468 Sabapathy, P.C., Devaraj S., Kathirvel P., 2017. Parthenium hysterophorus: low cost substrate for the production of polyhydroxyalkanoates. Curr Sci India 112:2106-2111 doi:10.18520/cs/v112/i10/2106-2111 Scheiner, D., 1976. Determination of ammonia and Kjeldahl nitrogen by indophenol method. Water Res 10:31-36 doi:Doi 10.1016/0043-1354(76)90154-8 Shimizu K. (1988) Substance and process for converting waste cooking oil into liquid soap. 4.839.089, Shogren, R.L., Petrovic Z., Liu Z.S., Erhan S.Z., 2004. Biodegradation behavior of some vegetable oil-based polymers. J Polym Environ 12:173-178 doi:DOI 10.1023/B:JOOE.0000038549.73769.7d 17

Jo

ur

na

lP

re

-p

ro of

Smith H., Winfield J., Thompson L. (2013) The market for biodiesel production from used cooking oils and fats, oils and greases in London. LRS Consultancy, Song, J.H., Jeon C.O., Choi M.H., Yoon S.C., Park W., 2008. Polyhydroxyalkanoate (PHA) production using waste vegetable oil by Pseudomonas sp. strain DR2. J Microbiol Biotechn 18:1408-1415 Tangy, A., Pulidindi I.N., Perkas N., Gedanken A., 2017. Continuous flow through a microwave oven for the large-scale production of biodiesel from waste cooking oil. Bioresource Technol 224:333-341 doi:10.1016/j.biortech.2016.10.068 Taniguchi, I., Kagotani K., Kimura Y., 2003. Microbial production of poly(hydroxyalkanoate)s from waste edible oils. Green Chem 5:545-548 doi:10.1039/b304800b Vastano, M., Casillo A., Corsaro M.M., Sannia G., Pezzella C., 2015. Production of medium chain length polyhydroxyalkanoates from waste oils by recombinant Escherichia coli. Eng Life Sci 15:700-709 doi:10.1002/elsc.201500022 Verlinden, R.A.J., Hill D.J., Kenward M.A., Williams C.D., Piotrowska-Seget Z., Radecka I.K., 2011. Production of polyhydroxyalkanoates from waste frying oil by Cupriavidus necator. Amb Express 1 doi:Artn 11 10.1186/2191-0855-1-11 Walsh, M., O'Connor K., Babu R., Woods T., Kenny S., 2015. Plant oils and products of their hydrolysis as substrates for polyhydroxyalkanoate synthesis. Chem Biochem Eng Q 29:123-133 doi:Doi 10.15255/Cabeq.2014.2252 Wang, Y., Ou S.Y., Liu P.Z., Xue F., Tang S.Z., 2006. Comparison of two different processes to synthesize biodiesel by waste cooking oil. J Mol Catal a-Chem 252:107-112 doi:10.1016/j.molcata.2006.02.047 Ward, P.G., Goff M., Donner M., Kaminsky W., O'Connor K.E., 2006. A two step chemobiotechnological conversion of polystyrene to a biodegradable thermoplastic. Environ Sci Technol 40:2433-2437 doi:10.1021/es0517668

18

Figure captions

Fig 1. Total Cell dry weight (g/l; ) and PHA (%CDW; ) of P. chlororaphis 555 when growing with waste cooking oil (WCO) pulse fed batches fermentations. (A) Feed strategy 1 (FS1) was designed based on monitoring of dissolved oxygen (DO) and used 220 g WCO/l, (B) FS2: 220 g WCO /l with phosphorus limitation, (C) FS3: 200 g WCO /l and phosphorus limitation, (D) FS4: 140 g WCO /l and

A

100 80 60 40 20 0

120

B

100 80 60 40 20 0

0

10

20

30

40

50

0

10

20

80 60 40 20 0

120

D

100 80 60 40 20 0

0

10

20

30

50

-p

C

100

40

40

50

0

10

20

30

Time (h)

Jo

ur

na

lP

Time (h)

re

120

30

Time (h) CDW (g/l), PHA (% CDW)

CDW (g/l), PHA (% CDW)

Time (h)

ro of

120

CDW (g/l), PHA (% CDW)

CDW (g/l), PHA (% CDW)

phosphorus limitation.

19

40

50

Table 1 Fatty acid content of waste cooking oil (WCO) used in this study and reported in literature. Fatty acids are represented as a % of total fatty acid content. Fatty acids Stearic

Oleic

Linoleic

Linolenic

(C16:0)

(C18:0)

(C18:1)

(C18:2)

(C18:3)

16.5

7.9

42.3

32.2

10

3

39

9

3

20

Other

Reference

n.db

0.7

This studya

48

n.db

n.db

Martino et al. 2014

37

50

0.1

n. b

Leung and Guo 2006

4.8

52.9

13.5

0.8

n.db

Cruz et al. 2015

13

4

33

43

4

n.db

Abidin et al.2013

8.2–8.6

4.0–4.7

27.2–30.6

55.1–57.6

0.3–2.2

0.5–0.6 Felizardo et al. 2006

Three independent samples of WCO were analysed in triplicate

b

-p

n.d. – not detected

ro of

a

Palmitic

Biomass* (g/l)

60 90 120

45.4 54 54.1

PHA (%CDW)

Biomass yield (g/g)

19.8 26.2 21.8

lP

WCO (g/l)

re

Table 2 Yield of biomass (g/g) and PHA (g/g) in batch fermentations using 60, 90 and 120 g WCO/l.

Jo

ur

na

*Total biomass is residual biomass and PHA combined

20

0.76 0.60 0.45

PHA yield (g/g)

0.14 0.15 0.09

Table 3 Growth data for P. chlororaphis 555 using different feeding strategies and concentrations of waste cooking oil Feed strategy

Biomass* (g/l)

Biomass yield (g/g)

PHA (g/l)

PHA volumetric productivity (g/l/h)

PHA yield (g/g)

FS1

62.10

0.28

7.14

0.15

0.03

FS2

82.45

0.37

17.15

0.36

0.08

FS3

79.40

0.40

10.70

0.22

0.05

13.87

0.29

0.11

ro of

FS4 73.00 0.52 *Total biomass is residual biomass and PHA combined

Table 4 Properties of PHA polymer extracted from P. chlororaphis 555 grown with waste cooking oil (WCO) under phosphorous limitation (entry 1) and comparison with PHA obtained from various

-p

substrates and bacterial strains (entries 2-5). Td – thermal degradation temperature, Tg – glass transition temperature, Tm – melting temperature, Mn – number, Mw –molecular weight. ND – not

re

determined.

Polydispersity (Mw/Mn)

Reference

Td (°C)

Tg (°C)

Tm (°C)

Mw (Da)

Mn (Da)

WCO

274±1.3

-64±1.4

-

18324±452

9874±2282

1.9

This work

290

-65

-

94796±188

52649±644

1.7

Walsh et al. 2015

289.4

-55.2

-

103652±390

59746±482

1.7

Walsh et al. 2015

45317±62

22954±975

2

Ruiz et al. 2019

36±1.2

30000

20000

1.5

Cruz et al. 2016

-

126700

70000

1.8

Ashby and Foglia 1998

HWCO$

270.6±1.6 -56.1±0.5 20.7±0.5 ND

-16±0.8

Jo

OODD&

na

HROFA**

ur

RO*

Soybean oil

lP

Substrate

ND

-45

*RO – rapeseed oil **HROFA – hydrolysed rapeseed oil fatty acids $HWCO – hydrolysed waste cooking oil &OODD – olive oil deodorizer distillate

21