WO2016039618A1

WO2016039618A1 - Bio-based crotonic acid production

Info

Publication number: WO2016039618A1
Application number: PCT/MY2015/050103
Authority: WO
Inventors: Hidayah ARIFFIN; Nur Falia Shazana MANJA FARID; Mohd Rahimi ZAKARIA@MAMAT; Mohd. Ali HASSAN
Original assignee: Universiti Putra Malaysia
Priority date: 2014-09-12
Filing date: 2015-09-13
Publication date: 2016-03-17

Abstract

The invention provides method for producing high selectivity trans-crotonic acid from bio-based resource wherein the steps comprise of treatment of poly(3-hydroxbutyrate) (PHB) biomass with mild alkali; and thermal degradation of the PHB biomass. Crotonic acid, a dehydrated monomer of PHB, can be obtained by thermal degradation under controlled temperature and retention time. Currently, crotonic acid is chemically synthesized via non-renewable resources. As an alternative, it can be produced biologically within cells in its hydrated polymer PHB form, and subsequently pyrolyzed to obtain crotonic acid. The invention also provides a method of producing crotonic acid by thermal degradation of wet or dry PHB biomass in the presence of magnesium compound. The present invention provides a method for treating PHB Biomass with low concentration of sodium hydroxide with molarity in between 0.05-0.1M.

Description

BIO-BASED CROTONIC ACID PRODUCTION

This present invention relates to a selective production of bio-based crotonic acid, more particularly to bio-based trans-crotonic acid

Various scientific and scholarly articles are referred to throughout the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains. Crotonic acid is an unsaturated carboxylic acid which exists in two geometric isomers; cis and trans-crotonic acid. This acid is the dehydrated monomer of PHB. Crotonic acid and its derivatives have many applications in industry such as components of hair styling products, paints, insecticides, softening agent for synthetic rubber, resin for coating and plasticizer (Singh et al., 2007, Journal of Molecular Catalysis A: Chemical, 266(1-2), 226–232; Babayig & Pulat, 2001, Journal of Applied Polymer Science,2690–2695; Ariffin et al, 2008, Polymer Degradation and Stability, 93(8), 1433–1439; Ariffin et al, 2010, Polymer Degradation and Stability, 95(8), 1375–1381 and Bassaid et al, 2008, Reactive and Functional Polymers, 68(2), 483–491).

Currently, crotonic acid is chemically synthesized from non-renewable resource which is petroleum. Chemical synthesis of crotonic acid involves many steps starting from steam cracking of natural gas liquids to produce ethylene, oxidation of ethylene to acetaldehyde, aldol condensation of acetaldehyde to acetaldol, dehydration of acetaldol to crotonaldehyde and lastly oxidation of crotonaldehyde to crotonic acid (Arpe, 2010 Industrial Organic Chemistry, Fifth edition Weinhein, Germany: Wiley-VCH Verlag GmbH Co. KGaA.). Two-stage of purification steps which are fractional distillation and crystallization from water are needed in order to obtain pure crotonic acid. However, during the crystallization from water, about one ton of highly contaminated effluent, which must be purified biologically, is formed per ton of crotonic acid. This is accompanied by about 1500 m3 (S.T.P) of air, from drying of the water- moist crotonic acid. Furthermore, the crystallization from water also causes product losses and lastly drying of the water-moist crotonic acid requires considerable amounts of energy (US Patent 4,918,225).

Since current industrial crotonic acid production is non-renewable and contributed to environmental pollution, new alternative has to be discovered for greener production of crotonic acid. Recent study showed that crotonic acid can be obtained through thermal degradation of PHB. Ariffin et al, 2008, Polymer Degradation and Stability, 93(8) reported that crotonic acid is a dehydrated monomer of PHB and it is the major decomposition product under suitable pyrolysis condition. Morikawa & Marchessault, 1981, Canadian Journal of Chemistry, 59(15), 2306–2313 showed that many impurities were present in the pyrolyzate of direct PHB cell pyrolysis. In order to obtain pure crotonic acid, purification must be done.

Another way to improve purity of crotonic acid is by doing recovery of the PHB containing-cell prior to pyrolysis. There are many methods of PHB extraction and purification available such as organic solvent extraction, chemical digestion, enzymatic digestion, mechanical method, but all of these methods are non-environmental friendly and expensive (Lee, 1996). The ideal method for PHB recovery should be chemically ‘green’ and inexpensive.

Hence, there is still a need for a method for production of crotonic acid that from bio-based material with an improved yield.

An aspect of the present invention is to provide a method for producing bio-based trans-crotonic acid comprises the steps of: providing poly(3-hydroxybutyrate) (PHB) biomass; pre-treatment of polyhydroxbutyrate (PHB) biomass with mild alkali; subjecting the pretreated PHB biomass to thermal degradation; and obtaining trans-crotonic acid;, wherein the crotonic acid yield ranges from 86%, and purity ranges from 97%.

Advantageously, the method employs biological synthesis and eco-friendly. The method of comprises the steps of producing crotonic acid by thermal degradation of PHB using specific degradation temperature.

Accordingly, the PHB polymer is obtainable from fermentation of bacteria. Additionally, the fermentation by PHB producer such as C. necator was carried out to get cell containing PHB in biomass form.

Accordingly, treatment of the PHB biomass with sodium hydroxide was conducted to remove non-polymeric cellular materials (NPCM). Advantageously, this step reduces impurities in the end product.

Accordingly, the treated PHB biomass is thermally degraded in the presence of magnesium compound to selectively degrade the polymer into trans-crotonic acid.

Advantageously, trans-crotonic acid yield is within the range of 75% to 86% with approximately 97% purity.

Fig.1

refers to TIC chromatograms of Dry PHB Biomass and Wet PHB Biomass.

Fig.2

refers to the TIC chromatograms of PHB Biomass, PHB extracted with Chloroform and PHB extracted with NaOH.

Fig.3

refers TEM images of PHA granules before and after treatment with sodium hydroxide (a) C.necator cell before treatment. (b) PHA granules of C.necator after NaOH treatment. Arrow indicates cell wall.

Fig.4

[FIG. 4] refers to the TIC chromatograms of PHB Biomass and PHB Biomass with addition of 5% Magnesium hydroxide.

Fig.5

[FIG. 5] refers to TIC chromatograms of PHB extracted with NaOH and PHB extracted with NaOH with addition of 5% Magnesium hydroxide.

A description of example embodiments of the invention follows. In general, the invention relates to a selective production of trans-crotonic acid with high purity via thermal degradation of PHB-containing biomass which has been treated with sodium hydroxide.

In some embodiments, the bacterial strain used is Cupriavidus necator, or other PHB producer such as Methylobacterium organophilum, Alcaligenes latus, and a recombinant E. coli. Carbon sources for fermentation are selected from a list of pure and simple substance such as glucose, sucrose, fructose, lactose, maltose and the like. Alternatively, complex sugar mixtures, fatty acids, vegetable oils and the like could also be used as fermentation feedstock (Chee et al., 2010, Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, 1395–1404). Fermentation is conducted with certain limitation to stimulate the accumulation of PHB granule. The limitations could be deficiency or limited supply of oxygen, or nutrients such as phosphorus or nitrogen.

In one embodiment, the PHB biomass obtained from fermentation process is used without undergoing drying process. After fermentation, PHB biomass is separated from the broth by centrifugation. Then, the biomass is washed with distilled water to remove the remaining broth before it is centrifuged again with similar condition as stated earlier. The biomass is then directly used for pyrolysis.

In some embodiment, the PHB biomass is treated with mild alkali for non-PHB cellular removal such as sodium hydroxide. Mild alkali (0.05 – 0.1M NaOH) is used and incubated at 4°C for 3 h with no agitation. After the NaOH treatment, the biomass pellet containing PHB is recovered by centrifugation at 8000 rpm for 10 minutes. The purification process is carried out by washing the biomass pellet with 1% (v/v) of ethanol (96%). Finally, the pellet was centrifuged and resuspended in distilled water for further washing prior to drying.

In some embodiment, magnesium compound is used as catalyst in order to selectively produce trans-crotonic acid. About 5 – 9 wt% of magnesium compound are added and mixed thoroughly before the pyrolysis reaction. The magnesium compound could assist in the reduction of degradation temperature as well as stimulating unzipping β- elimination by forming transition states which favor the degradation reaction. Therefore, by combining NaOH pretreatment step and addition of magnesium compound prior to PHB biomass pyrolysis, it is expected that high purity of trans-crotonic acid can be obtained.

In all embodiments, thermal degradation process is conducted using glass tube oven, vacuum heating equipment. Therefore, pyrolysis process is conducted in no or limited oxygen. The oven was heated from room temperature to 280-320°C. Volatile product is condensed in a cold trap.

In all embodiments, the thermal degradation products are measured and analyzed by using gas chromatography – mass spectroscopy (GC-MS).

PHB Samples	Pyrolyzates composition (%)
PHB Samples	Cis - Crotonic Acid	Trans - Crotonicacid	Oligomer	Impurities
PHB Biomass	5.01	57.12	35.39	2.48
PHB extracted with Chloroform	3.92	69.17	25.86	1.04
PHB extracted with NaOH	1.89	86.64	10.81	0.66
PHB Biomass + 5% MgOH₂	4.33	89.00	4.06	2.61
PHB extracted with NaOH + 5% MgOH₂	2.40	96.51	0.85	0.52
Wet PHB Biomass	4.98	58.97	33.65	2.40
Wet PHB Biomass + 5% MgOH₂	6.6	86.05	6.05	1.25

Crotonic acid is an important precursor for acrylic acid, butanol, propylene, maleic anhydride and advantageously the precursor is derived from bio-based resources. There are several other bio-based processes, such as from direct bacterial fermentation . However, no quantitative information was reported.

The present invention utilizes both biochemical and thermal approaches, surprisingly, these approaches produce higher yield and also high purity of trans-crotonic acid. Trans-crotonic acid is crotonic acid with trans orientation of functional group within its molecule. Most of the applications use trans- form of crotonic acid because of higher thermostability compared to cis- form of crotonic acid.

One of potential bio-based resources is palm-based waste which may be used as a raw material for bacterial fermentation of PHB. However, the bio-based resources for raw material is not limited only to palm based waste, other examples include beet molasses, date syrup, soya waste, malt waste, bagasse hydrolysates and saccharified potato waste.

The present technology is further depicted by the following examples.

Example 1

Production of crotonic acid by thermal degradation of wet and dried PHB biomass.

In this example, it is shown that crotonic acid can be obtained by pyrolysis of either wet or dry PHB biomass. The samples were heated using glass tube oven in a step-wise temperature increment. The oven was heated from room temperature to 200°C and the temperature was held for 30 min. Then, temperature was increased to 320°C and kept for 30 minutes. The vaporized pyrolyzates were condensed in cold trap and collected as white crystals. The white crystals were then analyzed by GC-MS.

Fig. 1 shows total ion chromatogram of both wet and dry biomass. As can be seen in Table 1, the results show that trans-crotonic acid is the major product (57.12% and 58.97% for dry PHB biomass and wet PHB biomass respectively) component in the pyrolyzate and there was no significant different in the composition for the two samples tested suggesting that drying process is not needed prior to pyrolysis.

FIG. 1 refers to TIC chromatograms of Dry PHB Biomass and Wet PHB Biomass.

Example 2

Production of crotonic acid by thermal degradation of untreated and sodium hydroxide-treated PHB biomass.

The following example compares the pyrolysis of untreated and NaOH-treated PHB biomass. For treated biomass, about 20 g/L of PHB biomass was treated with 0.05 M of sodium hydroxide prior to pyrolysis. The sodium hydroxide treatment is to remove non-PHB cellular materials thus increasing the PHB purity in the sample and eventually increasing the purity of crotonic acid in the pyrolyzates. Steps for pyrolysis were same as example 1.

Fig. 2 shows total ion chromatogram for both untreated and treated PHB biomass. As can be seen, treatment of NaOH was able to reduce the impurities present in the pyrolyzate and increase the purity of trans-crotonic acid from 57.12% to 86.64% (Table 1). NaOH treatment reduced the molar mass of the PHB (Table 2) and form crotonyl chain-end, which assisted in the degradation of PHB into crotonic acid. Furthermore, PHB treated with NaOH showed higher sodium content at 140ppm compared to chloroform-treated PHB (100 ppm). Residual sodium from NaOH treatment may act as catalyst and hence, helped in the formation of trans-crotonic acid. Figure 3 shows the reduction of cell wall composition after sodium hydroxide treatment.

Sample	Molar mass (Da)
Sample	M_n	M_w	M_w/M_n
PHB extracted with Chloroform	420,000	860,000	2.05
PHB extracted with NaOH	220,000	515,000	2.34

Mw: Weight average molar mass, Mn: Number average molar mass, Mw/Mn: Polydispersity in (Da)

Example 3

Production of crotonic acid by thermal degradation of dry PHB biomass with magnesium compound.

In this example, selective transformation or degradation of PHB into trans-crotonic acid is shown. Magnesium hydroxide (5 wt%) was added to the dry PHB biomass prior to pyrolysis at 290°C. As depicted in Fig. 4 and Table 1, trans-crotonic acid becomes the major product of the degradation (89%). There is no trimer detected and only small percentage of dimer is observed. Therefore, the results suggest that magnesium compound is able to selectively convert PHB into trans-crotonic acid.

Example 4

Production of crotonic acid by thermal degradation of NaOH-treated PHB biomass with magnesium compound.

In this example, the effect of sodium hydroxide treatment coupled with magnesium compound is examined. About 500 mg of sodium hydroxide-treated PHB biomass with 5 wt% of magnesium hydroxide was mixed thoroughly prior to pyrolysis at 290 °C for 30 min. Similar with example 3, the addition of magnesium compound was able to convert almost all dimer and trimer into monomer. Furthermore, the magnesium compound also able to convert most of the dimer into monomer, which consequently increases the overall purity of trans-crotonic acid up to 95% (Table 1 and Fig. 5).FIG. 5 refers to TIC chromatograms of PHB extracted with NaOH and PHB extracted with NaOH with addition of 5% Magnesium hydroxide.

As will be readily evident to those skilled in the art, this invention may easily be produced in other definite forms without leaving from its scope or essential characteristics. These embodiments are, therefore, to be considered as only illustrative and not restrictive, the scope of the invention being indicated by the claims rather than the former description.

Claims

A method of producing high selectivity trans-crotonic acid from bio-based resource, comprises the steps of:providing poly(3-hydroxybutyrate) (PHB) biomass; pre-treatment of polyhydroxbutyrate (PHB) biomass with mild alkali; subjecting the pretreated PHB biomass to thermal degradation; and obtaining trans-crotonic acid;, characterised in that;the crotonic acid yield ranges from 86%, and purity ranges from 97%.
The method of producing high selectivity trans-crotonic acid from bio-based resource according to Claim 1, wherein the pretreated PHB biomass for thermal degradation is selected from wet or dry pretreated PHB biomass.
The method of producing high selectivity trans-crotonic acid from bio-based resources according to preceding Claims, wherein the thermal degradation step is in the presence of a magnesium compound.
The method of producing high selectivity trans-crotonic acid from bio-based resources according to Claim 3, wherein the pretreated PHB biomass is physically mixed with the magnesium compound at weight percent of 5% – 9%.
The method of producing high selectivity trans-crotonic acid from bio-based resources according to Claim 3 or Claim 4, wherein the magnesium compound is selected from the list of Magnesium Oxide, Magnesium hydroxide and Magnesium chloride.
The method of producing high selectivity trans-crotonic acid from bio-based resources according to Claim 1, wherein the PHB biomass is treated with low concentration sodium hydroxide.
The method of producing high selectivity trans-crotonic acid from bio-based resources according to Claim 6 , wherein sodium hydroxide molarity is between 0.05 – 0.1M.
The method of producing high selectivity trans-crotonic acid from bio-based resources according to Claim 6, wherein treatment using mild alkaline reduces the molecular weight of PHB in the bacterial biomass and produces crotonyl-chain end which assisted in thermal degradation of PHB into crotonic acid.
The method of producing high selectivity trans-crotonic acid from bio-based resources according to Claim 6, wherein traces of Na+ ion left after mild alkaline treatment assisted in the degradation of polymer to crotonic acid.
A product produced from the method according to the preceding claims.