High-Purity Fatty Acid n-Octyl Esters from Housefly (Musca

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High-purity fatty acid n-octyl esters from housefly (Musca domestica) larval lipid, a potential new biolubricant source Zi-Zhe Cai, Sheng-qing Wu, Guo Sun, Yi Niu, Dong Zheng, Shi-yi Peng, Wen-zhe Yang, Yong Wang, and Depo Yang Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.7b01996 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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Energy & Fuels

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High-purity fatty acid n-octyl esters from housefly (Musca domestica L.) larval

2

lipid, a potential new biolubricant source

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Zi-zhe Caia, Sheng-qing Wua, Guo Sunb, Yi Niua, Dong Zhenga, Shi-yi Penga,

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Wen-zhe Yangc, Yong Wangb,*, De-po Yanga,*

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a School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou,

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Guangdong Province, People’s Republic of China,

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b Dept. of Food Science & Engineering, Guangdong Saskatchewan Oilseed Joint Lab.,

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Jinan University, Guangzhou, Guangdong Province, People’s Republic of China,

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c Zhongshan Unicare Natural Medicine Co, Ltd, Zhongshan, Guangdong Province,

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People’s Republic of China.

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* Corresponding author.

12

Abstract:

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With the traditional lipid feedstock cost constituting 70% of the total biolubricant

14

production, it is urgent to find a new lipid source biodiesel and biolubricant industry.

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With elevated acid value (61.8 mg KOH/g) of housefly larval lipid, converting free

16

fatty acid (FFA) into biolubricant could serve as a value-added way to the larvae

17

industry, other than pretreated with acid-catalyzed esterification for biodiesel

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production. This study developed a method of producing high-purity fatty acid n-octyl

19

esters (FAOEs) from housefly (Musca domestica L.) larva. The housefly larva free

20

fatty acid (HLFFA) from the larva lipids was obtained through wipe-film short path

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distillation. FAOEs were produced by esterifying n-octanol with HLFFAs (catalyzed

1

ACS Paragon Plus Environment

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by benzenesulfonic acid under the following conditions: catalyst loading, 2 wt.%;

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molar ratio of n-octanol to FFA, 3:1; temperature, 100 °C; time, 2 h). Excess

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was effectively removed by high vacuum (80 Pa) distillation at 90 °C. The unreacted

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FFA were neutralized using demethylated crude glycerin, which is the by-product of

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biodiesel production. A practical method of producing high purity fatty acid n-octyl

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esters (99.19 wt.%) derived from housefly larva lipid is developed and the product

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could serve as replacement of certain low viscosity mineral lubricants such as liquid

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paraffin, polyalphaolefin 6 and SN500.

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Keywords: Musca domestica L.; biolubricant; fatty acid n-octyl ester; esterification;

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crude glycerin neutralization.

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1. Introduction

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A lubricant or lube is a liquid that provides functions of reducing the friction

34

between moving surfaces, improving efficiency, reducing wear, preventing surface

35

corrosion, removing wear particles , and dissipating heat from wear surfaces 1. The

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estimated global biolubricant demand reaches 42.1 million metric tons by 2017 at an

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annual increase of 2% 2, however, the mineral-based lubricant occupies about 98.8%

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base oil market share 3. One of the critical obstacle preventing sustainable bio-based

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lubricant from broader application is the feedstock cost. As its primarily feedstock is

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virgin vegetable oil (such as palm oil

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higher than conventional mineral-based oils 6. In developing countries which are not

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self-sufficient in edible oils 7,8, such lipid source would not be suitable because of the

4

and rapeseed oil 5), the cost is four to five

2


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inadequate supply and high price of edible oils. Therefore, finding a low-cost and

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non-food feedstock for biolubricant production is urgently needed.

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Insect larva is a renewable source of protein and lipid. Both laboratory scale

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research (black soldier fly, Hermetia illucens L.9; flesh fly, Boettcherisca peregrine10;

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blow fly Chrysomya megacephala8; and Zophobas morio11) and pilot level production

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(blow fly, Chrysomya megacephala12) have utilized larva lipid for biodiesel

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production. With large amounts of municipal waste (nearly 1.3 billion tons worldwide)

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and livestock manure (1.9 billion tons in China) generated annually, considerable

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amount of lipid could be produced by rearing insect larva on such wastes 13. By now,

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insect lipid used as biodiesel feedstock have drawback of higher acid value. Our

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previous research has demonstrated that additional heating energy (nearly tripled) is

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required for heating acylglycerols (during esterification process of free fatty acid,

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FFA) and methanol recovery step, if acid-catalyzed pre-esterification is applied

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The acid value of lipid from reported insect species usually is higher than virginal

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vegetable oil (2-21 mg KOH/g)

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FFA into FAME via aforementioned two-step method.

14

.

9,11,15

, and it is not energy efficient converting larval

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The housefly Musca. domestica L. (Diptera: Muscidae) is a well-known

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cosmopolitan holometabolous insect with features like: can be fed with various source

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of waste materials

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reproductive rate. Compared with black solider flies (H. illucens) housefly has shorter

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larvae period (4-5 days v.s. 33 days) and longer adult longevity (over a month v.s. 15

16

; contains high lipid content (around 20%)

3


15

; faster and

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days) 17, and has similar lipid content (about 20%). Besides, after lipid extraction, the

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feed residues can be used as a fertiliser, and the defatted larvae proteins can be used

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livestock feed

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domestica L. larvae possess highly active lipase, and their fatty acid content readily

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increases during fresh larva processing in response to unfavourable conditions

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Instead of reducing the unfavourable FFA, the alternative is to converting fatty acid

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value-added product such as biolubricant. Through wipe-film distillation (small scale)

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or deodorizer (industry scale), FFA could be separated from larval lipid and served as

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low-cost feedstock for biolubricant.

18

, making it a value-added product for housefly lipid industry. M.

12

.

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Produced from esterification with another low-cost feedstock, n-octanol, which is

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the cheapest among long-chain alcohols (C8–C14) 19, fatty acid n-octyl esters (FAOEs)

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has applications (as low viscosity fluid) like modern two-stroke engines 20, cold-metal

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rolling 21, coolants for metal machinery 22 and drilling fluids for marine oil industry 23.

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Such low viscosity lubricant has advantages of low toxicity, biodegradable, miscible

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with various additives, oxidative stable and devoid of undesirable impurities

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However, there are drawbacks in reported FAOE production studies (Table 1): a)

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longer reaction time is required to achieve high conversion (above 98%) when using

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acid

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p-toluenesulfonic acid

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addition of alcohol and catalyst when using enzymatic esterification

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conversion rate is relatively low; c) unclear explanation on n-octanol removal; d)

or

supported

catalyst

(nitrogen-doped

26

, and amberlyst 15H

19

reduced

graphene

oxide

24

.

25

,

with reaction time of 4-24 h); b)

4


27–29

and

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insufficient details on removing unreacted FFA; e) comprehensive lubricity evaluation

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remains to be evaluated (e.g. water separability, non-seizure load, and weld load). In

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our previous research, 98.6% esterification rate was achieved under conditions: 3:1

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molar ratio of 2-ethyl hexanol to FFA, 2 h, 2 wt.% loading of BSA (benzenesulfonic

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acid)

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n-octanol removal requires high vacuum (near 100 Pa) other than methanol (5000 Pa),

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With larger processing capacity than Kugelrohr evaporator, n-octanol can be removed

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completely (residual n-octanol is lower than 0.5%) through wipe-film distillation,

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which is promising for massive production. Lubricant product with FFA content

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than 2% (approximately 4 mg KOH/g) is not acceptable to the ASTM and EU

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for biolubricant (< 0.6 mg KOH/g), which might cause possible damage of filtration

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system

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evaporation nor high-vacuum molecular distillation can separate FFAs and FAOEs

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because their actual evaporation pressures are close (preliminary experiment). FFA in

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the crude FAOE product can be effectively neutralized by alkali refining, which is

100

30

. According to the finding of Moser et al. using Kugelrohr evaporator

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,

22

. For the unreacted FFA in the esterification product, neither low-pressure

developed in the edible oil refining industry 30.

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In the present study, high-purity FAOEs were produced using FFA from housefly

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(M. domestica) larvae lipid. The FFA fraction from housefly larval lipids (housefly

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larva free fatty acid, HLFFA) was obtained via wipe-film short path distillation and

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followed by benzenesulfonic acid (BSA)-catalyzed esterification with n-octanol.

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Excess n-octanol in the esterification product was removed through wipe-film short

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path distillation. The residual fatty acids in the product were neutralized with the

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by-product, demethylated crude glycerin (see Figure 1). The tribological properties of

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the produced housefly larval FAOEs were tested for the first time. The present study

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demonstrates a practical method to produce high-purity biolubricant (FAOEs).

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Developing an added-value product is a promising solution for biodiesel production

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because it can extend the production supply and supplement the biodiesel or edible

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refining industry, from which the by-products (FFAs) can be used as feedstock for

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FAOE production.

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2. Materials and methods

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2.1 Materials

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M. domestica larvae were obtained from Zhongshan Unicare Natural Medicine,

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Zhangshan, Guangdong. The larvae were identified by Prof. Hong Pang from the

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State Key Laboratory of Biocontrol and Institute of Entomology, Sun Yat-sen

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University. Analytical grade n-octanol, n-hexane, sodium hydroxide, benzenesulfonic

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acid were purchased from Damao Chemical Reagent Factory (Tianjin, China). The

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esterification and neutralization apparatus consisted of an oil bath with a magnetic

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stirrer, a digital temperature controller fixed in the oil bath and a 250 mL single-neck

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round-bottom flask. Kitchen waste was collected from the canteen of Zhongshan

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Unicare Natural Medicine, Zhangshan, Guangdong.

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The neutralization agent (for section 2.7), demethylated crude glycerin, was

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produced from transesterification of housefly larval acylglycerols (HLAG) with

6


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methanol, and characterized (supporting information). The base value of

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crude glycerin was 50.4 mg KOH/g.

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2.2 Larva inoculation

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Kitchen waste was collected 2 h before housefly larva inoculation. The kitchen

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waste was rinsed with hot water (80 °C) and cooled down. The resulting feeds for the

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larvae were spread evenly to a depth of approximately 5 cm in each plastic tank (60

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cm × 20 cm × 15 cm). The inoculum density was 8000 larvae/kg feed. After

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inoculation, the plastic tanks were maintained at a temperature of 28 °C with a

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relative humidity of 75% and a photoperiod of 12:12 (L:D).

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After 5 days, the mixture containing the feeds and larvae was blended with dry

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wheat bran to reduce the moisture and viscosity of the medium. The mixture was

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poured into a larva collector equipped with screens and two slopes. The obtained

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larvae were mostly free of feed residue and the total mass of fresh larvae was

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recorded. 2.3 Processing of fresh larvae and extraction of housefly larval oil

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For each patch, 200 g of extracted fresh larvae were introduced into a sealed glass

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box (5 cm × 20 cm × 20 cm) and placed in a 50 °C oven for 5 h. The preheated larvae

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were incapacitated with boiling water (100 °C) and then oven dried at 80 °C for 6 h.

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10 kg dried housefly larvae were crushed to a 60 mesh powder by a powder

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machine. Oil extraction was executed using a single-neck round-bottom reactor

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equipped with condenser, the addition of n-hexane is 60 kg (mass ratio of n-hexane to

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larva powder: 6:1) for 5 h at room temperature (25 °C). Subsequently, the solvent

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filtrated via vacuum filtration, then it was removed under reduced pressure using a

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rotary evaporator at 50 °C. The lipid content (%) was calculated by dividing the mass

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extract lipid with mass of larvae powder.

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2.4 Separation of HLFFAs

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HLFFA was separated by a wipe-film short path device (MD 80; Guangdong

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Hanwei Co., Ltd.) with a falling film evaporator (area: 0.1 m2) and an internal

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condenser (area: 0.05 m2). A jacketed glass vessel with a flow-regulating valve was

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used to introduce oil to the distillation equipment. The vacuum system was composed

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of a mechanical pump and a diffusion pump. The separation parameters are as follows:

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temperature, 120 °C; feeding rate, 200 mL/h; evaporator vacuum, 0.5–1.0 Pa (using

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both mechanical pump and diffusion pump); condenser temperature, 50 °C; and

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feeding temperature, 80 °C. In each patch, 1 kg of extracted larval lipid was subjected.

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The distillate (mostly HLFFAs) and the residue (mostly housefly larva acylglycerols,

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HLAGs) were collected. The HLFFAs would be used as feedstock for the subsequent

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esterification with n-octanol (biolubricant production), whereas the HLAGs would be

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transesterified with methanol (biodiesel production).

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2.5 Esterification of HLFFAs with n-octanol

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The rate of FFA esterification (ER) into n-octanol esters depends on several

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factors, such as reaction temperature, reaction time, catalyst loading, stirring rate and

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n-octanol content. Single-factor experiments were performed to evaluate the

8


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parameters affecting the ER. The catalyst ranged from 0 wt.% to 4 wt.% (based on the

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HLFFA mass) with an interval of 1 wt.%. The n-octanol-to-HLFFA molar ratio

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ranged from 1:1 to 9:1 with an interval of 2:1. The reaction time ranged from 1 h to 5

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h with an interval of 1 h. The reaction temperature ranged from 80 °C to 160 °C with

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an interval of 20 °C. The stirring rate was fixed (300 rpm).

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HLFFA sample (50.0 g), n-octanol and benzenesulfonic acid were introduced to a

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round-bottom flask equipped with a reflux condenser. The reaction was conducted in

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an oil bath under aforementioned reaction conditions at a vacuum level of 5000 Pa.

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Water was removed while n-octanol was refluxed throughout each reaction. After

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each experiment, the mixture was washed with hot water (80 °C) twice to remove

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benzenesulfonic acid residues and were saved for further analysis. Each experiment

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was performed in triplicate, and the means and standard deviation (means ± SD) were

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reported.

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The ER of HLFFA to FAOE was calculated using the following equation:

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ER = 1 - AVest/AV0,

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where AVest and AVo refer to the AV of the esterified product and initial AV of

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the reactant mixture (HLFFA with n-octanol), respectively.

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2.6 n-Octanol removal via wipe-film distillation

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The esterification product under optimum condition was washed with hot water

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(80 °C) twice to remove the acid catalyst and subjected for n-octanol removal

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processing. The excess n-octanol was removed using the aforementioned distillation

9


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device (MD 80; Guangdong Hanwei Co., Ltd.) operated without the diffusion pump

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(In this case, the device served as high vacuum distillation). As the vacuum is

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determined by the structure of the distillation apparatus, the vacuum degree was fixed

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at 80 Pa. Feeding rate was fixed at 400 mL/h, as faster feeding rate would raise

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problem of pressure stabilization due to the evaporated n-octanol. A single-factor

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experiment of evaporating temperature was conducted under conditions: vacuum

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degree, 80 Pa; condenser temperature, 20 °C; and feeding temperature, 80 °C. The

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evaporator temperatures ranged from 60 °C to 120 °C with an interval of 10 °C. The

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distillate was mostly n-octanol, and it was collected for repeated use. The residue

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enriched with FAOEs was weighed. The AV of each removal experiment was

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determined. The effectiveness of each experiment was expressed as octanol removal

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efficiency (RE) and FAOE recovery rate (RR), which were calculated via 1H-NMR

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analysis (section 3.2). Concentrations (C, wt.%) of FAOE, n-octanol in the samples

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were determined. Each experiment was performed in triplicate, and the means and

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standard deviation (means ± SD) were reported.

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The removal efficiency (RE) of n-octanol was calculated by equation:

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REn-octanol (wt.%) = (C’-Cn-octanol)/ C’

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Where C’, Cn-octanol are the n-octanol concentration (wt.%) before and after each

207

208

distillation, respectively.

The recovery rate (RR) of FAOE was calculated by equation:

10


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RRFAOE (wt.%) = (Ccrude FAOE × mcrude FAOE - CFAOE × m FAOE)/ Ccrude FAOE × mcrude FAOE

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Where Ccrude FAOE, CFAOE, mcrude FAOE, m FAOE are FAOE concentration (wt.%) of

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crude FAOE, FAOE concentration (wt.%) of FAOE after n-octanol removal process,

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mass (g) of crude FAOE subjected to wipe film distillation, and mass (g) of FAOE

214

after n-octanol removal process, respectively.

215 216

2.7 Neutralization of residual FFA with crude glycerin from biodiesel production

217

After n-octanol removal, the n-octanol-removed product (under optimum

218

condition) was collected. On the basis of our preliminary experiment on crude

219

glycerin neutralization (Figure S2), 5 g of the demethylated crude glycerin 5 g and 50

220

g of n-octanol-removed product (under optimum condition) were introduced to a

221

single-neck round-bottom flask. The mixture was heated in an oil bath at 80 °C for 20

222

min with an agitation rate of 300 rpm. After the reaction, flask contents were

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separated into two layers in a separatory funnel. The upper layer was clear orange

224

(FAOEs product), which was then washed with hot water (80 °C) twice. The washed

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upper layer was dried in a vacuum oven for 2 h at 60 °C, then the mass of product is

226

weight (g) and yield (wt.%) is recorded (mass of product divided by mass of crude

227

FAOE used for neutralization).

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3. Analysis 11


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3.1 Acid value determination

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Acid values of sample from crude larval lipid, esterification, n-octanol removal

231

process, and neutralization were determined through titration with KOH in

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accordance with AOCS Method Ca 5a-40.

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3.2 Nuclear Magnetic Resonance (NMR) Spectroscopy.

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NMR spectra was obtained with a Bruker AM-400 spectrometer at 25 °C. NMR

235

data collection and analysis were conducted with MestReNova 11.0.3 software

236

(Mestrelab Research S.L.).

237

For analysis of n-octanol removal products and the final product, deuterated

238

chloroform (NMR grade, Aladdin Industrial Corporation, Shanghai, China) and

239

3,4,5-trichloropyridine (TCP) (NMR grade, Aladdin Industrial Corporation, Shanghai,

240

China) were used as solvent and internal standard, respectively. 4.83 mg internal

241

standard and 81.4 mg sample were dissolved in 500 µL solvent. The molecular weight

242

of FAOE, n-octanol, TCP are 371.5, 130.2, and 182.43 g/mol, respectively. The triplet

243

at 4.07 ppm (2H, t, CH2O) refer to FAOEs

244

-CH2OH) refers to n-octanol. The singlet at 8.53 ppm (4H, s) refers to TCP (internal

245

standard). All above mentioned peaks were integrated for quantitative analysis.

32

and the triplet at 3.65 ppm (2H, t,

246

The FAOE concentration were calculated by equation:

247

CFAOE (%) =(mTCP/msample) × (IFAOE/ITCP) × (MFAOE/MTCP)× (HTCP/HFAOE)

248

The n-octanol concentration was calculated by equation:

12


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Cn-octanol (%) =(mTCP/msample) × (In-octanol/ITCP) × (Mn-octanol/MTCP)× (HTCP/Hn-octanol)

250

where m denotes the weight in g, M the molecular weight in g/mol, H the marked

251

proton number of each compound and I the NMR integral area.

252

3.3 FAOE composition via GC-MS

253

The FAOE composition of the final product was analyzed by using a Finnigan

254

GC-MS system equipped with a DB-WAX capillary column (50 m × 0.25 mm i.d.,

255

0.25 mm film thickness) (Trace-Ultra/DSQ; Thermo Electron Co., Waltham, US)

256

with helium as carrier gas at a flow rate of 1.0 mL/min and a pressure of 100 kPa. The

257

split ratio was 1:30. The oven temperature was set to 210 °C, and the holding time

258

was 21 min. Electron ionisation mode (ionisation energy of 70 eV) was used for GC–

259

MS detection. The temperature of the injector and MS transfer was 250 °C. The

260

scanning mass range selected was 30–650 m/z. The sample volume of each injection

261

was 1.0 µL. Data were managed with Xcalibur software with the NIST mass spectra

262

library database. The relative contents of each FAOE species were calculated using

263

the area normalisation method, and the results were reported as percentages of the

264

total peak area. The determination was performed in triplicate, and the means and

265

standard deviation (means ± SD) were reported.

266

3.4 Physical and chemical properties of the final product

267

The final product was analyzed by Guangdong Inspection and Quarantine

268

Technology Center (Guangzhou, PRC). The testing methods for viscosity, flash point, 13


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base value, AV, density, copper strip corrosion, pour point, water separability and

270

sulphur content are listed in Table 3. Lubricity test, including wear scar diameter

271

(WSD), last non-seizure load (PB value) and weld load (PD value), was conducted

272

using a four-ball friction and wear-testing machine according to Chinese standard

273

GB/T3142-1982, which was similar with ASTM D2783. The steel ball was made of

274

bearing steel GCr15 (hardness of 60 HRC), which was ultrasonically cleaned before

275

and after testing with petroleum ether and acetone. The WSD of stationary balls was

276

measured using a Nikon PFX optical microscope installed with a Nikon F-301 CCD

277

camera. The PB value was determined in accordance with ASTM D2783. The rotating

278

speed was 1450 rpm, and a series of tests with 10−s duration at increasing load was

279

conducted. The PB value was determined when the corresponding WSD was not more

280

than 5% above the compensation line described in ASTMD-2783. The PD value was

281

the lowest applied load (1450 rpm, 10−s) when rotating ball welds to stationary balls.

282

Free glycerin and total glycerin content was analyzed according to ASTM standard

283

D6584-13. The soap content was determined by AOCS method Cc 17-95.

284

4. Results and discussion

285

4.1 Processing of fresh larvae and lipid extraction.

286

Approximately 109 ± 2.8 g of fresh larvae were obtained from each kg of feeds,

287

thus 23.98 ± 1.1 g dried larvae could be harvested from each kg of feeds. After

288

solvent extraction, 23.2 kg larval lipid could be obtained from 100 kg dried larvae.

14


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Energy & Fuels

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The acid value of larval lipid was 61.8 mg KOH/g. (FFA content 27.9 wt.%). The

290

AVs of the distillate (HLFFA) and residue (HLAG) after wipe-film distillation were

291

215.97 and 0.56 mg KOH/g, respectively.

292

Owing to the nature of insect lipid metabolism, the acylglcyerols were inevitably

293

hydrolysed during mass production: The larvae would response to unfavorable living

294

environment as the temperature elevated by increased population density during

295

extraction process. Arrese et al. 33 suggested that the lipase in the insect fat body is an

296

active phospholipase A1. In addition, the lipid hydrolysis process could be initiated

297

by the AKH (adipokinetic hormone) which excretes from corpora cardiac of insects.

298

Similar to the active temperature of commercialised phospholipase A1, the active

299

temperature range was 30–50 °C. The lipase exhibits both triacylglycerol-lipase and

300

phospholipid-lipase abilities, indicating that the lipase hydrolysed the membrane and

301

had access to the stored acylglycerols. Among larval incapacitation methods such as

302

sun drying, freezing and refrigeration, heating at 50 °C without ventilation greatly

303

increases the lipid AV of larvae in a short period of time without adding any

304

hydrolysis agent. The elevated temperature had two possible effects on the larval lipid

305

hydrolysis: a) It stimulated the larval response towards unfavourable temperatures and

306

b) altered the membrane structure of the lipid drop in the fat body, allowing the lipase

307

to access the triacylglycerols. In addition, in the preliminary experiment at 50 °C with

308

ventilation, the AV increased to only 25.1 mg KOH/g because larvae were dehydrated.

309

When processed without ventilation, the AV of the larval lipid increased to 61.8 mg

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Energy & Fuels

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310

KOH/g as less water was evaporated (as a reactant of lipid hydrolysis). As mentioned

311

in the introduction, most of the produced FAOEs involve high-purity fatty acids

312

feedstock. Thus, producing such self-hydrolyzed lipid is efficient and economic

313

compared with those involving lipase for lipid hydrolysis 19. Housefly larvae fed with

314

various waste materials exhibit high adaptability and high resistance to diseases. Thus,

315

the massive production of such high-AV lipids can greatly reduce the cost of

316

biolubricant production, and various lipid products like various kinds of fatty acid

317

esters can be produced with such lipid feedstock.

318

4.2 Esterification of HLFFAs with n-octanol

319

As the catalyst loading increased from 0.5 wt.% to 2 wt.%, the ER (esterification

320

rate) was elevated from 78.7% to approximately 93% before it reached a plateau

321

(Figure 2a.). High conversion (over 90%) can be achieved within a short reaction time

322

(2 h vs. 6 h) and BSA requires fewer amounts of n-octanol compared with

323

p-toluenesulfonic acid

324

n-octanol in esterification and subsequence recovery step is conserved. In room

325

temperature (25°C), BSA forms white deliquescent sheet crystals or a white waxy

326

solid, with melting point and boiling point of 44 °C and 137 °C 34. Thus it is easy to

327

handle, transfer, and storage

328

catalyst melts to catalyze esterification and could be recovered for recycle usage after

329

cooling 34. Compared with other acid catalysts (e.g. heteropoly acid, sulfated zironcia,

330

zeolite, and other supported catalyst)

26

(3:1 vs. 10:1). In addition, the heating energy for both

35

. Under elevated reaction temperature (100 °C), the

25

, BSA has advantages like widely available, 16


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Energy & Fuels

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and no preparation is required. In addition, unlike other substituents (e.g.

332

3-amino-4-methylbenzenesulfonic acid; 4-amino-3-methylbenzenesulfonic acid),

333

BSA is reported 90% biodegradable 36, and it can be recovered by high vacuum.

334

As displayed in Figure 2b, the initial molar ratio of n-octanol to FFA was 1:1, and

335

the reaction was conducted at 100 °C for 2 h with a catalyst loading of 2 wt.%. The

336

ER increased from 85.1% to 93.1% when the molar ratio of n-octanol to FFA reached

337

3:1. The ER then slightly decreased to 92.8% as the molar ratio continued to increase

338

to 9:1. The decrease was probably due to the dilution of BSA catalyst. Compared with

339

enzymatic esterification

340

greatly reduced, with higher conversion rate (94% vs. 85%). With regard to energy

341

efficiency and feedstock cost, a 3:1 molar ratio of n-octanol to FFA is recommended.

19,27

, the amount of n-octanol required for esterification was

342

As illustrated in Figure 2c, molar ratio of n-octanol (3:1) to FFA is based on the

343

result of above experiment, and the ER was around 80% initially because the lower

344

temperature decreases intermolecular contact of each reactant. The ER increased to

345

approximately 92% and then reached a plateau as the temperature exceeded 100 °C.

346

In theory, high temperatures can promote the reaction equivalence towards the

347

positive direction. However, this condition could induce the evaporation of reactants,

348

especially n-octanol, and thus could reduce the probability of intermolecular contact.

349

In addition, high reaction temperatures might lead to the generation of darkly

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350

coloured esterification products found by reported n-octyl ester production

351

such instance, a reaction temperature of 100 °C is recommended.

Page 18 of 40

19,37

. In

352

As described in Figure 2d, the ER was 87% after 1 h of reaction. It increased to

353

approximately 93% and became stable as the reaction time was prolonged. Therefore,

354

a reaction time of 2 h is recommended, whereas reported FAOE synthesis used

355

prolonger reaction time to achieve conversion rate of above 90% (4–6 h for other

356

types of acid catalyst and 6–10 h for enzymatic esterification) 19,26,28.

357

On the basis of the single-factor experiments above, the optimum reaction

358

conditions were as follows: temperature, 100 °C; catalyst loading, 2 wt.%; molar ratio

359

of n-octanol to FFA, 3:1; and time, 2 h. The ER under such conditions was 95.1%.

360

4.3 n-Octanol removal via wipe-film distillation

361

As shown in Figure 3, as the temperature was elevated from 60 °C to 100 °C, the

362

RE of n-octanol increased sharply from 30.0 % to 98.8 % and then reached a plateau

363

above 99 %. This result indicates that the wipe-film distillation was effective and

364

efficient for n-octanol removal. Such results were also consistent with a previous

365

study

366

and 65 Pa. In addition, RR of FAOEs slightly decreased from 96.7 % to 91.3 % when

367

the temperature exceeded 90 °C, indicating that trace amount of FAOEs could have

368

been distilled as colour change of distillate was observed (transparent into yellow). A

369

high RR of FAOEs can be achieved using high feeding rate (400 ml/h), as both

26

where excess octanol was removed through Kugelrohr evaporator at 120 °C

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distillation time and the possibility of FAOE being distilled can be decreased. The

371

decrease of RR at higher temperature also indicates that the optimum distillation

372

temperatures for FFA and FAOE are close (approximately 90 °C). Thus, it is

373

impossible to separate FFAs from FAOEs using high vacuum distillation. The AV

374

slightly increased from 5 mg KOH/g to approximately 7.0 mg KOH/g and then

375

decreased to 5.8 mg KOH/g when the temperature exceeded 90 °C. The increased AV

376

was due to elevation of FFA concentration (n-octanol was removed). The

377

subsequence AV decrease was observed because more FFA is distilled at higher

378

temperature. Considering above, the optimum evaporator temperature is 90 °C with

379

n-octanol removal rate of 98.9 wt.%, FAOE recovery rate of 95.6 wt.%, and acid

380

value of 6.69 mg KOH/g.

381

4.4 FAOEs neutralization by crude glycerin

382

Under the optimum conditions of n-octanol removal, the AV of the crude FAOE

383

residue was 6.69 mg KOH/g. As suggested in previous studies, one drawback of

384

acid-catalyzed esterification is that the AV of the product is greater than 2.0 mg

385

KOH/g, which does not satisfy the AV requirement of the ASTM and EU standards

386

(

High-Purity Fatty Acid n-Octyl Esters from Housefly (Musca

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