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Improved fermentability of pretreated glycerol enhanced bioconversion of 1,3-propanediol Jian Ping Tan, Zhao Kang Tee, Wan Nor Roslam Wan Isahak, Byung Hong Kim, Ahmad Jaril Asis, and Jamaliah Md Jahim Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b02268 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018

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Industrial & Engineering Chemistry Research

Improved fermentability of pretreated glycerol enhanced bioconversion of 1,3-propanediol Jian Ping Tana, Zhao Kang Teea, Wan Nor Roslam Wan Isahaka,b, Byung Hong Kimc, Ahmad Jaril Asisd, Jamaliah Md Jahima,b* KEYWORDS: 1,3-propanediol; C. butyricum; glycerol; microfiltration; pre-treatment

ABSTRACT

The potential of biodiesel-derived glycerol to be valorised into 1,3-propanediol (1,3-PDO) was hindered by the impurities contained therein. This study introduced a straightforward and effective pre-treatment process through microfiltration to remove free fatty acids (FFAs) that could inhibit the fermentation which resulted in remarkable high productivity in batch fermentation. This approach appreciably reduces the cost of pretreatment as compared to previously reported acidification, solvent extraction, electrodialysis, activated carbon adsorption and ion-exchange resins. From the fermentation results, it was found a high productivity of 1,3PDO of productivity of 2.16 gL-1h-1 was attained when 20 g/L pre-treated glycerol was used as the carbon source, an improvement of 62.4% compared with fermentation with crude glycerol using locally isolated strain Clostridium butyricum JKT37, and among the highest compared to previous reports. These results suggested that, with a simple microfiltration pre-treatment, the utility of the resultant biodiesel-derived glycerol in bio-production of 1,3-PDO is comparable to

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that of pure glycerol. The physical and chemical properties of the biodiesel-derived glycerol were characterised in accordance with American Oil Chemists' Society (AOCS), International Organization for Standardization (ISO), and American Society for Testing and Materials (ASTM) standard methods, before and after the pre-treatment. The sample of crude glycerol comprised of 75 wt % glycerol, 15% soap, 8% moisture, 0.8% of methanol and less than 5% of ash. FFAs generated from crude glycerol were removed completely by microfiltration.

1.0 Introduction The increase of fuel usage associated with mass transportation and construction has led to correspondingly greater demand for renewable biofuels,1 among which are crude oil alternatives. In this regard, accordingly, the focus is on building more biodiesel plants with lower operating costs. One shortcoming, nonetheless, is the excessive generation of glycerol, the by-product of biodiesel production. It is estimated that biodiesel processing plants generate one-tenth mass of glycerol from every unit mass of biodiesel during transesterification,2,3,4 culminating in copious organic waste.5 Companies encountered difficulties in overcoming the costs of purification of crude glycerol in order not to exceed the disposal limits set by authorities.6 Glycerol, a by-product of biodiesel production, is obtained through the transesterification of methanol and vegetable oil.7 Glycerol has a promising potential to serve as the carbon source in the production of 1,3-propanediol.8,9 Crude glycerol consists of water, remaining methanol, free fatty acids (FFAs), methyl esters of fatty acids and glycerides, inorganic salts and ash.10 However, the final type and composition of impurities in crude glycerol are determined by the source of feedstock in the biodiesel production. Few studies have elucidated the sources of oil in biodiesel production, such as palm,11 soy bean,12 sunflower,13 and jatropha.14 Among all, Asad ur


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et al. have shown the composition of crude glycerol with 30% glycerol, 50% methanol, 13% soap, 2% water, remaining 2-3% of inorganic salts and matter organic non-glycerol (MONG).13 However, characterisation by Thomson and He revealed that glycerol content ranged from 60 to 70 wt% in different types of feedstock of mustard, rapeseed, canola, crambe, soybean, and waste cooking oils.15 Glycerol can be categorised into different grades, according to the glycerol content. In general, crude glycerol contained 60–80% glycerol whereas purified glycerol in contains, by percentage composition, at least 99.1% glycerol.16 As compared with glycerol, the term ‘glycerine’ is more commonly used in commercial synthesis of glycerol of a technical or pure grade with at least 99.5% purity. Besides, the specifications of glycerine in pharmaceuticals and food have to comply with the United State Pharmacopeia and Food Chemical Codex grades of 99.5%.17 Accordingly, the direct utilization of crude glycerol to substitute for glycerine in industrial applications, without the removal of impurities, has not been not feasible.18 The current market price of crude glycerine is around RM850 per tonne as compared to the price of biodiesel at RM2389, or RM2.05 per litre.19 Instead of selling off to epichlorohydrin industry in Thailand and China, crude glycerol could be utilised to increase Malaysia revenue. A techno-economic analysis conducted by Posada et al.20 has shown that PDO is the most profitable biorefinery using raw glycerol, with the ratio of sale price to production cost at 1.83. This value is much higher when compared to scenarios in other chemical (hydrogen, acrolein, 1,2-propanediol and glycerol purification) and biochemical (ethanol, lactic acid, succinic acid, propionic acid, and poly-3-hydroxybutyrate) industries. Moreover, there are few current key manufacturers of PDO using crude glycerol as the raw materials, such as Metabolic Explorer, Glory Biomaterial, and Zouping Mingxing. It is expected the market of PDO will gain a


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compound annual growth rate at 10.4% from 2014 to 202121 due to its largest application in polytrimethylene terephthalate (PTT) industry.22 The price of PDO is estimated to exceed RM15,000 per tonne, which is 18 times higher than the price of crude glycerol by year 2019.23 The purification of glycerol to glycerine includes separation steps such as acid wash, removal of methanol, filtration, and vacuum distillation.24 Acid wash is usually the primary step in which strong acids are added to convert the remaining catalyst and soaps in the crude glycerol. The use of sulphuric acid for repeated acidification of crude glycerol to pH 1–6 increases the yield of glycerol from waste used-oil methyl ester plant.25 The use of 5.85% phosphoric acid was also proposed to increase the glycerol-rich volume from 40% to 70% at pH 5–6.26 This is followed by methanol removal to recover the raw material in biodiesel production. Owing to the economic advantage and detrimental environmental concern of excess methanol emission, an evaporator or flash unit necessitated in industrial purification. The purity of crude glycerol can reach up to 85% after methanol is removed. The third step of glycerol purification involves filtration. EET Corporation has patented a glycerol-purification technology named High Efficiency Electro-Pressure Membrane (HEEPM™), in which the unit configuration includes nanofiltration and reverse osmosis.24 Though membrane technology required less energy to operate, its utility in industrial practice is still unfavourable due to persistent concerns on membrane fouling and durability.27 Nevertheless, vacuum distillation is the most established technique to industrially purify crude glycerol,28 affording production of glycerol of 96.6% purity at 120–126ºC and 0.4 to 0.04 mbar.26 Conversely, pre-treatment methods have been introduced to reduce impurities in biodiesel-derived glycerol. The quality of pre-treated glycerol need only be sufficiently high to


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be applicable for other uses such as fermentation. Asad Ur et al. (2008)13 had performed a series of pre-treatments that included acidification with phosphoric acid, followed by solvent extraction and finally distillation to remove FFAs.13 Nevertheless, Moon et al. were unable to remove MONG using hexane, due to lack of formation of a separation layer during extraction.30 For removal of high sodium salt content from crude glycerol, Jensen et al. conducted electrodialysis and were able to remove 98% of the salts.31 The removal of toxic compounds during glycerol fermentation can be achieved by the utilisation of activated carbon adsorption.32 Lately, a superior pre-treatment of crude glycerol by ion-exchange resins was discovered, demonstrating effectiveness in removing salts by up to 33% and MONG by up to 55%,33 though it has yet to be commercialised. The conversion of partially pre-treated crude glycerol into more value-added products can be achieved through fermentation. Among the most extensive research is the use of glycerol for 1,3-propanediol (1,3-PDO) production through anaerobic fermentation using Clostridium sp.34,35 Some studies show that high contents of impurities such as FFAs, methanol, and salt content in crude glycerol may inhibit the growth of the Clostridium sp.36,37

Research by

Chatzifragkou et al. (2010) on inhibition of C. butyricum VPI 1718 found the following: while the presence of crude glycerol with NaCl content exceeding 4.5% and of oleic acid at 2 wt % inhibited bacterial growth, that of alcohol content up to 10 wt% did not impair it.36 However, Moon et al. reported that methanol content at 6.67 wt % was likely to inhibit the fermentation by C. butyricum DSM 15410.6,30 Based on specifications provided by the manufacturer, crude glycerol contains soap (max. 25%), methanol (max. 10%), and oil as ester (max. 10%). Therefore, in the production of 1,3-PDO, it is essential to consider reducing these impurities to an acceptable range which do not hinder fermentation.


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While efficient purification of glycerol cannot depend on a single process to remove all impurities within the crude glycerol, pre-treatment will be a more feasible way. This study focuses on pre-treatment involving microfiltration as an effective approach to remove impurities in biodiesel-derived glycerol. Physico-chemical properties of crude and pre-treated glycerol are characterised for comparisons. In addition, the fermentabilities of crude and pre-treated glycerol, with pure glycerol as control, were evaluated in batch fermentation that conducted in a 3.6-L bioreactor.

2.0

Materials and Methods

2.1

Pre-treatment of glycerol

The crude glycerol used herein was obtained from Sime Darby Biodiesel Sdn. Bhd. Selangor, Carey Island, Malaysia. Biodiesel is the product of transesterified palm olein and methanol, with glycerol as a by-product. Crude glycerol is not suitable to be used in fermentation media due to its high pH and insolubility in water at room temperature. For this reason, a series of pretreatment was introduced to eliminate its impurities. As a step to remove methanol, the crude glycerol was pre-heated at 60 ºC in a water bath until it liquefied and turned dark brown colour. As it was heated, part of the methanol content would be eliminated due to its volatility. To the liquid crude glycerol thus prepared was added 37 % hydrochloric acid until a pH of 4.5 to 5.0 was attained. The experiment was conducted in a ventilated fume hood. In the acidification of crude glycerol, excess soaps in the mixture were removed and converted into free fatty acids (FFAs). At this stage, crude glycerol appeared to be a yellowish liquid, with blackish oil droplets


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on the surface of liquid. For the liquid has cooled down, the black oils formed clumps and solidified on the surface of liquid. The mixture was filtered through a rayon-based filter paper with a vacuum pump. The pH of the filtrate was maintained at pH 7 using 2M potassium hydroxide solution before it was stored under 4 ºC as pre-treated glycerol. 2.2

Local isolated strain

The microorganism used in the fermentation was C. butyricum JKT37 (NCBI accession number: KU513553). The microbe was locally isolated from wastewater treatment plant of palm oil mill effluent (POME) which were collected from Sime Darby East Oil Mill, Selangor, Malaysia. The isolation method was previously described as in Tee et al. (2017).35 2.3

Characterisation of glycerol samples

The glycerol prepared was characterised as regards its physical properties such as density and viscosity. The density of glycerol was measured by using density meter Anton Paar DMA 5000. The instrument was calibrated with deionized water for accuracy up to ± 0.0001 before use. Besides, its viscosity was tested using a Brookfield DV II+ Pro viscometer with the LV-2 spindle in accordance to ASTM D 4878-08.38 As for the chemical properties, the pH of glycerol was determined by dissolving 1 g of it in 50 mL of deionized water and measuring it at room temperature using Sartorius pH meter PB-10. The chemical compositions of crude and pre-treated glycerol were analysed as regards glycerol content, moisture content, methanol concentration, free fatty acid content, soap content, total ash and glyceride content. Crude glycerol was heated at 60 ºC in a water bath before being dissolved for further analysis. The glycerol content was determined using the titrimetric method according


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to the AOCS Official Method Ea 6-9439 and ISO 2879-1975 (E).40 A total of 50 mL of 5% (w/v) glycerol sample was diluted with water to about 250 mL. Adjustment with sodium hydroxide solution yielded a mixture with pH 7.9, to which 50 mL of sodium metaperoxide solution was added. The solution was kept in the dark for 30 minutes, afterwards further mixed with 10 mL of 1,2-ethanediol, and allowed to stand for another 20 minutes. To this was added 5 mL of sodium formate solution, and finally titration against sodium hydroxide solution followed. A blank test without the glycerol was carried out simultaneously. The glycerol content was calculated as a percentage by mass, by the formula as follows:

Glycerol content, % =

where

(V − V ) × N × 0.0921 × 100 m

V1 = volume in mL of standard sodium hydroxide solution used for glycerol

V2 = volume in mL of standard sodium hydroxide solution used for blank N = normality of sodium hydroxide, i.e. 0.125 m = mass in grams of the test portion The water content of glycerol was determined using DL38 Volumetric Karl Fischer Titrator (Mettler Toledo) in accordance to the AOCS Official Method Ea 8-58.41 The underlying principle is that water was titrated with iodine-methanol solution in the presence of a base. The end point of titration was determined by a bipotentiometric method, which involved the abrupt voltage between two electrodes. For the estimation of methanol concentration, 1 g of glycerol was dissolved in 100 mL deionized water and tested

by high performance liquid

chromatography (Thermo Scientific UltiMate 3000), equipped with Phenomenex RoA 300 mm ×


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7.8 mm column and refractive index detector (RID). The mobile phase used was 5mN H2SO4, which eluted isocratically at a flow rate of 0.6 mL/min and temperature 60ºC. The mass percentage of methanol in the sample was calculated from a standard plot of chromatogram area versus methanol concentration. The content of free fatty acids (FFAs) was analysed according to the AOCS Official Method Ca 5a-40.42 To hot neutralised ethanol and 2 mL of phenolphthalein was added 28.2 ± 0.2 g of glycerol sample. The mixture was then titrated with standardised sodium hydroxide until the appearance of the first permanent pink as that of the blank. The percentage of FFAs as palmitic acid was calculated as follows:

Free fatty acids as palmitic, % =

mL of alkali × M × 25.6 mass, g of test portion

Where M = normality of sodium hydroxide solution The soap content in the glycerol sample was determined using the titrimetric method as published by ISO 1615-1976(E).43 The test portion consisted of a mixture of 40 ± 0.1 grams of glycerol sample and 100 mL of warm (40ºC) acetone, with bromophenol blue solution as the pH indicator. The mixture was titrated with hydrochloric acid solution with stirring until the colour changed from blue to yellowish green. The alkalinity of glycerol can be expressed by the formula as follows:

Alkalinity, milliequivalents per 100g = 10

Where

V m

V = volume in mL, standard 0.1N hydrochloric acid used for titration


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m = mass in grams of the test portion The determination of ash content in glycerol was done with the reference of ISO 6884:2008.44 A total of 10 grams of weighed sample was burnt at 550 ºC for 4 hours with the addition of hydrogen peroxide, until carbon-free ash was obtained. The ash content, expressed as a percentage by mass, is given by the formula as follows:

Percentage of ash, % =

m − m m/

where, m0 = mass in grams of the glycerol sample m1 = mass in grams of the empty crucible m2= mass in grams of the crucible and ash Glycerides content were determined according to ASTM D6584-10a.45 The glycerol sample was injected into Agilent 6890 Series GC System with flame ionization detector, equipped with Phenomenex Zebron™ ZB-5HT Inferno capillary column. Gas chromatography (GC) conditions were as follows: detector temperature, 360°C; initial oven temperature, 800°C; initial holding time, 0 min; ramping rate, first ramp 20°C/min and second ramp 10°C/min; final temperature, 350°C; final holding time, 20 min; carrier gas, helium; flow rate, 0.8 mL/min; column pressure, 11.38 psi; and injection volume, 1.0 mL. 2.4

Batch glycerol fermentations


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The composition of the fermentation media are (per litre of water): K2HPO4, 3.4 g; KH2PO4, 1.3 g; (NH4)2SO4, 2.0 g; MgSO4·7H2O, 0.2 g; CaCl2·2H2O, 0.02 g; yeast extract, 1.0 g; Lcysteine HCl, 0.5 g; 5 g/L resazurin solution, 1 mL; Fe solution, 1 mL; and trace element solution, 2 mL. The Fe solution consisted of FeSO4.5H2O, 5 g/L and 37% hydrochloric acid, 4 mL/L. The trace solution comprised (per one litre of water) ZnCl2, 70 mg; MnCl2·4H2O, 100 mg; H3BO3, 60 mg;

CoCl2·2H2O, 200 mg;

CuCl2·2H2O, 20 mg;

NiCl2·6H2O, 20 mg;

Na2MoO4·2H2O, 40 mg. Samples of pure-grade, crude and pre-treated glycerol with concentration 20 g/L were respectively added to the medium as the sole carbon source for the fermentation, which was conducted in a 3.6-L bench-top Labfors 5 bioreactor (Infors HT, Switzerland) with a 1-L working volume. For crude glycerol fermentation, the medium was prepared with heated and liquefied crude glycerol beforehand. The pH of fermentation was adjusted by adding 4M potassium hydroxide solution. The inoculum used in the experiment was taken during the exponential phase and 10% v/v was transferred into the fermentation mixture with sterile syringes. The bioreactor had been sparged with 99.9% pure nitrogen until the dissolved oxygen level reached zero before the inoculum was added. The fermentation experiments were carried out at 33ºC with an agitation speed of 200 rpm. Samples were taken for analysis at hourly intervals. 2.5

Analytical Methods

All the liquid samples of fermentation products consisting 1,3-PDO, acetic acid and butyric acid were measured using UltiMate 3000 high performance liquid chromatography, HPLC (Thermo Scientific, USA), equipped with Phenomenex RoA 300 mm × 7.8 mm column and refractive index detector (RID). A 5 mN H2SO4 solution was used as the mobile phase, which eluted isocratically at a flow rate of 0.6 mL/min. The column temperature was set at 60ºC and the


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compounds detected by the refractive index detector at 40ºC. A sample measuring 1 mL was filtered beforehand through the 0.22 µm membrane pore size into a sterile HPLC vial.46 Biomass concentration was analysed using the standard plot of dry cell weight versus optical density at 600 nm. 3.0

Results and Discussion

3.1

Pre-treatment of crude glycerol

This study suggests three main processes in the pre-treatment, namely acidification, microfiltration and neutralisation. A simple process flow diagram to illustrate the pre-treatment processes is shown in Figure 1, in which every stream is labelled from 1 to 8, with Stream 1 denoting crude glycerol and Stream 8 pre-treated glycerol. In addition, the physical appearances of glycerol throughout the treatments are indicated, with labels corresponding to stream numbers. Crude glycerol, the mixture of the un-reacted oil, methanol and some reduced catalyst in the form of soaps, appeared to be yellowish-brown solid at room temperature. It had a distinctive alcoholic smell and appeared oily when in contact. On the contrary, acidified crude glycerol appeared to be a yellowish liquid, with blackish oil droplets on its surface. Microfiltration removed the black oil clumps, which then formed the fatty matter in Stream 5. Pre-treated glycerol appeared physically clear and slight yellowish due to the removal of impurities.


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Pre-treated Glycerol

Figure 1. Process flow diagram for glycerol pre-treatment The composition of crude glycerol (Stream 1) was determined through the characterisation of glycerol (see Table 2). Notwithstanding its high glycerol content, impurities in the crude glycerol included 9.52 % (w/w) soap, 0.76 % (w/w) methanol, 7.51 % (w/w) moisture, 3.40 % (w/w) inorganic ash, and 0.75 % (w/w) free fatty acids. Due to high fatty acid content, crude glycerol formed a solid at room temperature. The crude glycerol was heated at a mild temperature of 60 ºC, with the twofold aims of improving its quality by partially removing methanol vapours and of liquefying the mixture and using it as the fermentation medium.


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The heated crude glycerol was found to be unsuitable for fermentation due to its high soap content with a concomittant increase of pH to 11.72. Hence, acidification was warranted to convert the excessive soap into free fatty acids and salts under controlled heating. Traces of the added catalyst were found to react with hydrochloric acid to produce methanol and salts. The chemical reactions occurred in acidification are shown in Equations 1 and 2.

0123 + 567 → 99: + ;267

…(1)

;2

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