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Application of (R)-3-Hydroxyalkanoate Methyl Esters Derived from Microbial Polyhydroxyalkanoates as Novel Biofuels Xiaojun Zhang,†,‡ Rongcong Luo,†,‡ Zhen Wang,‡ Yuan Deng,‡ and Guo-Qiang Chen*,‡,§ Multidisciplinary Research Center, Shantou University, Shantou, Guangdong 515063, China, and Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, China Received December 9, 2008; Revised Manuscript Received January 19, 2009
Microbial polyhydroxyalkanoates (PHA) were proposed for the first time as a new type of biofuel. In this paper, poly-R-3-hydroxybutyrate (PHB) and medium chain length PHA (mcl PHA) were, respectively, esterified to become R-3-hydroxybutyrate methyl ester (3HBME) and medium chain length hydroxyalkanoate methyl ester (3HAME) via acid-catalyzed hydrolysis. The recovery percentages of 3HBME and 3HAME were 52 and 65%, respectively. The purities of 3HBME and 3HAME were 97 and 96%, respectively. Combustion heats of 3HBME, 3HAME, ethanol, n-propanol, n-butanol, 0# diesel, 90# gasoline, and 3HBME-based and 3HAME-based blended fuels were investigated and compared, respectively. It was found that 3HBME and 3HAME had combustion heats valuing 20 and 30 KJ/g, respectively. Ethanol has a combustion heat of 27 KJ/g, while addition of 10% 3HBME or 3HAME enhanced the combustion heat of ethanol to 30 and 35 KJ/g, respectively. The addition of 3HBME or 3HAME into n-propanol and n-butanol led to a slight reduction of their combustion heats. Combustion heats of blended fuels 3HBME/diesel or 3HBME/gasoline and of 3HAME/diesel or 3HAME/gasoline were lower than that of the pure diesel or gasoline. It was roughly estimated that the production cost of PHA-based biofuels should be around US$1200 per ton.
Introduction Polyhydroxyalkanoates (PHA) are diverse biopolyesters synthesized by as many bacteria as carbon and energy reserved materials under unbalanced growth conditions.1,2 Many efforts have been made to commercially use PHA as an environmentally friendly bioplastics.3 However, high production cost for PHA must be effectively addressed before PHA can be used in a large scale. One of the possibilities to reduce PHA production cost is the use of waste substrates for fermentation, as almost 30% of total PHA production cost is attributed to the carbon source.4 Worldwide, a great amount of excess activated sludge is generated daily.5 Handling, treatment, and ultimate disposal of the excess sludge accounts for 40∼60% of the total operational cost of an activated sludge treatment plant.6 Therefore, excess sludge management has been directed for moving toward reutilization of sludge as useful resources, such as the use of excess sludge for PHA production7-9 and for phosphorus removal.10 On the other hand, volatile fatty acids (VFA) are one of the main intermediates in the anaerobic sludge fermentative liquid,10,11 and long chain length fatty acids (LCA) are commonly found when lipids are available from household wastewater,12 which are the most suitable substrates for microbial PHA production. Under alkaline conditions, the yield of VFA and LCA were enhanced significantly from excess sludge anaerobic fermentation.11-13 PHA produced from wastewater has been well documented.9,14 Many monomers, such as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), and 3-hydroxy* To whom correspondence should be addressed. Tel.: +86-754-2901186. Fax: +86-754-2901175. E-mail:
[email protected]. † Both authors contributed equally to this paper. ‡ Shantou University. § Tsinghua University.
dodecanoate (3HDD), have been found to be in the PHA structures either as homopolymers or copolymers.2 Poly-R-3hydroxybutyrate (PHB) belongs to short chain length PHA (scl PHA) containing only 3HB monomers. In contrast, medium chain length PHA (mcl PHA) are copolymers containing 3HHx, 3HO, 3HD, and 3HDD monomers. These 3-hydroxyalkanoates (3HA) monomers link each other with ester bond linkage formed with hydroxyl group (-OH) of one monomer and carboxyl (-COOH) group of the other monomer via the catalysis of various bacterial PHA synthases.15 Although PHB is the most common PHA in the activated sludge, mcl PHA were also found.16 As an energy carrier, PHA is to provide energy to cellular activities under starvation conditions.17 Many methods including biotransformation, fermentation using genetic engineering strains, and acid, alkali, or enzyme-catalyzed hydrolysis of PHA have been developed to obtain PHA monomers.4,19 Using these methods, 3HA and its derivatives, such as 3HA methyl esters (3HAME), 3HA ethyl ester, and so on, were obtained.20 Renewable fuels such as biofuels derived from biomass are rapidly developed,21,22 which include bioethanol, biomethanol, vegetable oils, biodiesel, biogas, bio-oils, biochar, and biohydrogen. Traditional biofuels including bioethanol and biodiesel have several advantages: (a) biofuels are easily produced from common biomass sources, (b) biofuels have a considerable environmental friendliness, and (c) biofuels are biodegradable and contribute to the world’s sustainability.21,22 However, the production of biofuels has led to the rising cost of staple foods.23 When considering the production process, chemical structures and role as energy carrier, it is easily found that 3HA esters are similar to those of biofuels, particularly similar to biodiesel that is methyl esters of long-chain fatty acids. In addition, 3HA esters are very different from petroleum. Petroleum contains very low oxygen yet high nitrogen and sulfur contents, which can lead to environmental pollutions upon burning. In contrast, 3HA
10.1021/bm801424e CCC: $40.75 2009 American Chemical Society Published on Web 02/27/2009
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Table 1. GC Results of 3HBME and 3HAME monomer composition (mol %) samples
a
yield (%)
purity (%)
65 52
96 ( 2.32 97 ( 1.71
c
mcl PHA 3HAMEd 3HBMEe
b
3HHx
3HOb
3HDb
3HDDb
3.0 ( 0.1 1.85 ( 1.2
22.9 ( 0.3 29.06 ( 0.8
33.2 ( 1.0 33.11 ( 2.1
40.9 ( 1.4 35.98 ( 0.1
a Yield: recovery percentage yield. It was determined from the ratio of the mass of 3-hydroxybutyrate methyl ester (3HBME) or 3-hydroxyalkanoate methyl ester (3HAME) to the original PHA mass. b 3HHx: 3-hydroxyhexanoate in mcl PHA; 3-hydroxyhexanoate methyl ester in 3HAME. 3HO: 3-hydroxyotanoate in mcl PHA; 3-hydroxyotanoate methyl ester in 3HAME. 3HD: 3-hydroxydecanoate in mcl PHA; 3-hydroxydecanoate methyl ester in 3HAME. 3HDD: 3-hydroxydodecanoate in mcl PHA; 3-hydroxydodecanoate methyl ester in 3HAME. c mcl PHA: medium chain length polyhydroxyalkanoate. d 3HAME: medium chain length hydroxyalkanoate methyl ester. e 3HBME: 3-hydroxybutyrate methyl ester.
esters have high oxygen content yet without nitrogen and sulfur. Because it has been commonly recognized that oxygenated additives can decrease exhaust smoke for diesel, reduce the ignition delays, and shorten the combustion duration and engine emissions of smoke, HC and CO can also be reduced. However, it is difficult to completely fuel diesel engines directly with oxygenated additives like 3HA esters due to its low cetane number and high latent heat of vaporization. Consequently, the development of 3HA esters as sustainable fuel or fuel additives may contribute to the diversification of biofuel or fuel additive market. Meanwhile, the exploitation of 3HA esters as biofuels may provide new insight into present “food versus fuel” controversy. The applications of PHA in the plastic industry and medical areas have been extensively studied, while no attention has been paid to turn PHA into biofuels at present. Therefore, the aim of this study was to evaluate the potential of 3HA methyl esters as a new type of biofuel.
Materials and Methods Materials. PHB was purchased from Jiangsu Lantian Group (Jiangsu China). The number average molecular weight (Mn) was 350000 and polydispersity (Mw/Mn) was 1.75, as revealed by gel permeation chromatography (GPC). Mcl PHA was synthesized in our laboratory using fadA and fadB genes knockout mutant Pseudomonas putida KTOY06 with lauric acid as a carbon source. The fermentation conditions and the resulting mcl PHA had been described.24,25 The following 3HA monomers were found in the synthesized mcl PHA with different compositions. They were 3 mol % 3-hydroxyhexanoate (3HHx), 23 mol % 3-hydroxyoctanoate (3HO), 33 mol % 3-hydroxydecanoate (3HD), and 41 mol % 3-hydroxydodecanoate (3HDD). Preparation of 3-Hydroxyalkanoate Methyl Esters (3HAME). 3-Hydroxybutyrate methyl ester (3HBME) and 3HAME were obtained via acid-catalyzed hydrolysis of PHB and mcl PHA.20,26 The following are the details: 15 g PHA were dissolved in 200 mL of chloroform or acetyl butyrate. After the addition of 200 mL of acidic methanol (15% (v/v) H2SO4 in methanol), the mixture was refluxed at 100 °C for 60 h. The mixture was subsequently cooled to room temperature, after which, 40 mL of saturated NaCl solution was added. After vigorous stirring for 10 min, the organic and water phase were separated. The water phase was subsequently extracted three times with 50 mL of chloroform each time. After drying the combined organic phase using Na2SO4 and evaporating the solvent under vacuum, a viscous and ester-like transparent liquid was obtained. This is the 3HBME or a mixture of various 3HAME. GC and FTIR Study of 3HA Methyl Esters (3HAME). Gas chromatography (GC) (Agilent 6890, U.S.A.) was used to determine the purity of the hydrolytic 3HA methyl esters. Benzoic acid was used as an internal standard. Detailed operation and calculation methods were the same as described previously.27 Fourier transform infrared spectroscopy (FTIR; Nicolet IR 200, Thermo Electron Corporation, U.S.A.) was used to investigate the chemical structures of 3HA methyl esters. A total of 32 scans at a
resolution of 4 cm-1 were recorded for each sample. The liquid 3HAME were smeared on the KBr plate and then subjected to analysis. Combustion Heats of 3HBME, 3HAME, and Blends. The combustion heats of the 3HBME, 3HAME, and their blends with ethanol, gasoline, diesel, and so on, were determined using oxygenbomb combustion calorimeter (BH-IIIS, Nanda Wanhe Group, Nanjing, China). Combustion heats defined with this oxygen-bomb combustion calorimeter were isometric combustion heats (Qv), which is determined in constant volume. The equation of the relationship between a temperature change and a combustion heat is
C × ∆t)Qv×S - Qn × (m1 - m2)
(1)
In this equation, C is the value of a constant heat capacity of the calorimeter, which is determined with a standard substance such as benzoic acid [C7H6O2, (Qv ) -3226.7 KJ/mol]; ∆t is temperature change of 3 L of water surrounding the oxygen-bomb before and after the burning of the substances; Qv is the isometric combustion heat; S is the mass of the substances; Qn is the combustion heat of the fuse wire; nickel wire with a combustion heat of -1.4 KJ/g is used as the fuse in this study; and (m1 - m2) is the mass change of the nickel wire before and after the burning process. Structures, principle, and operation method of the calorimeter has been described in previous studies.28
Results and Discussions GC and FTIR Study of Hydrolytic 3HAME. To prepare 3HAME, recovery yield and purity are two important factors that should be considered. The recovery yield was determined with the ratio of recovered 3HAME to the original PHA. The purity was detected with gas chromatography (GC; Table 1). In addition, molar composition changes of 3HHx, 3HO, 3HD, and 3HDD in mcl PHA before and after the hydrolysis were also compared with GC results (Table 1). Recovery yield for both 3HBME and 3HAME samples were 52 and 65%, respectively. On the other hand, the purities of both 3HBME and 3HAME were above 95%. According to GC analysis, molar compositions of 3HAME including methyl esters of 3HHx, 3HO, 3HD, and 3HDD were similar with those corresponding 3-hydroxyalkanoates monomer contents found in the mcl PHA (Table 1). The expected 3HAME fit the corresponding peaks very well in FT-IR spectra (Figure 1). For both samples, groups of -OH, -CdO-, -CsOsC-, and -CsH- appear at 3454 cm-1, 1740 cm-1, 1260∼1160 cm-1, and 2960∼2860 cm-1, respectively. Additionally, the major difference of the FTIR spectra between 3HBME and 3HAME is in the band of -C-H- located at 2960∼2860 cm-1 (Figure 1). The characteristic peak located in the 1620∼1680 cm-1, which is the double bond (CdC) of crotonic acid, was not observed (Figure 1), implying the elimination of crotonic acid from 3HBME after distillation. The major difference in the FTIR spectra of 3HBME and 3HAME
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Table 3. Combustion Heats of 3HBME, 3HAME, and Its Blends with Ethanol, n-Propanol, or n-Butanol samplesa
CHb (KJ/g)
3HAME/ethanol 1/9 2/8 3/7 4/6
34.84 ( 0.026 33.68 ( 0.28 28.41 ( 0.33 31.87 ( 0.11
3HAME/n-propanol 1/9 2/8 3/7 4/6
35.00 ( 0.68 34.34 ( 0.28 29.36 ( 0.26 27.19 ( 0.08
3HAME/n-butanol
Figure 1. FTIR spectra of 3HAME and 3HBME: 3HBME ) 3-hydroxybutyrate methyl ester; 3HAME ) medium chain length hydroxyalkanoate methyl ester. Table 2. Combustion Heats of 3HBME, 3HAME, and Its Blends with Ethanol, 0# Diesel, and 90# Gasoline samplesa
CHb (KJ/g)
3HAME/ethanol 1/9 2/8 3/7 4/6
34.84 ( 0.026 33.68 ( 0.28 28.41 ( 0.33 31.87 ( 0.11
3HAME/0# diesel 1/9 2/8 3/7 4/6
47.40 ( 0.86 45.78 ( 0.28 49.12 ( 0.38 41.08 ( 0.17
3HAME/90# gasoline 1/9 2/8 3/7 4/6
53.26 ( 0.15 50.37 ( 0.11 47.80 ( 0.078 45.56 ( 0.34
samplesa
CHb (KJ/g)
1/9 2/8 3/7 4/6
35.52 ( 0.096 31.88 ( 0.066 34.67 ( 0.39 34.56 ( 0.47
samplesa
CHb (KJ/g)
3HBME/ethanol 1/9 2/8 3/7 4/6
30.36 ( 0.39 29.83 ( 0.03 30.35 ( 0.36 30.48 ( 0.52
3HBME/n-propanol 1/9 2/8 3/7 4/6
36.85 ( 0.16 35.00 ( 0.27 35.39 ( 0.03 31.79 ( 0.42
3HBME/n-butanol 1/9 2/8 3/7 4/6
39.67 ( 0.30 39.88 ( 0.61 36.28 ( 0.42 33.42 ( 0.51
a Samples: (1) 3HBME ) 3-hydroxybutyrate methyl ester; 3HAME ) medium chain length hydroxyalkanoate methyl ester. (2) The ratio of 3HBME or 3HAME based blended fuels is the mass ratio. (3) The combustion heat (KJ/g) of pure 3HBME, 3HAME, ethanol, n-propanol, and n-butanol is like the following: 3HBME ) 19.87 ( 0.26; 3HAME ) 30.33 ( 0.09; ethanol ) 27.13 ( 0.26; n-propanol ) 37.37 ( 0.09; n-butanol ) 39.09 ( 0.028. b C H: Combustion Heat.
3HBME/ethanol 1/9 2/8 3/7 4/6
30.36 ( 0.39 29.83 ( 0.03 30.35 ( 0.36 30.48 ( 0.52
3HBME/0# diesel 1/9 2/8 3/7 4/6
49.52 ( 0.60 45.15 ( 0.84 43.87 ( 0.31 38.43 ( 0.72
3HBME/90# gasoline 1/9 2/8 3/7 4/6
44.39 ( 0.14 44.54 ( 0.51 42.67 ( 0.23 38.75 ( 0.46
a Samples: (1) 3HBME ) 3-hydroxybutyrate methyl ester; 3HAME ) medium chain length hydroxyalkanoate methyl ester. (2) The mass ratio of 3HBME or 3HAME based blended fuels. (3) The combustion heats (KJ/ g) of pure 3HBME, 3HAME, ethanol, 0# diesel, and 90# gasoline are: 3HBME ) 19.87 ( 0.26; 3HAME ) 30.33 ( 0.09; ethanol ) 27.13 ( 0.26; 0# diesel ) 50.10 ( 0.864; 90# gasoline ) 52.19 ( 0.69. b CH: combustion heat.
was in the -C-H- area. Due to the longer side chain of 3HAME, the vibrancy of -C-H- in 3HAME led to a split of two obvious peaks in FTIR spectra. Combustion Heats of 3HBME, 3HAME, and their Blends with Other Fuels. Combustion heat is one of the most important criteria used to evaluate the quality of a fuel. The combustion heat of 3HBME, 3HAME, and their blends with ethanol, 0# diesel, and 90# gasoline were studied and compared (Table 2). It was clearly found that 3HBME had the lowest combustion heat valuing 20 KJ/g among the 3HAME, and the 3HAME used in this study had a combustion heat of 30 KJ/g. Though 3HAME had a lower combustion heat than that of 0# diesel valuing 50 KJ/g and 90# gasoline valuing 52 KJ/g, it is much higher than that of ethanol, which is only 27 KJ/g. When blending 3HBME with ethanol, 0# diesel, or 90# gasoline, it was found that the combustion heat of ethanol was improved approximately 18-30% (Table 2). In contrast, the blending of 3HBME with 0# diesel or 90# gasoline reduced the
combustion heats of diesel and 90# gasoline. However, the blended fuels of 3HBME/0# diesel and 3HBME/90# gasoline still maintained a combustion heat valuing 38-50 KJ/g. Combustion heat changes for the 3HAME based blended fuels were similar with those of 3HBME based blends. Interestingly, although 3HAME has almost twice the combustion heat value of 3HBME, its blending with ethanol did not provide much improvement for the heat (Table 2). With the increase of 3HBME or 3HAME contents, combustion heats of both 3HBME and 3HAME blended with ethanol did not change significantly. In contrast, 3HBME or 3HAME blended with 0# diesel showed a decreasing trend from 49 to 38 KJ/g or 47 to 41 KJ/g, respectively, with increasing 3HBME or 3HAME contents (Table 2). The addition of 3HBME or 3HAME enhanced the combustion heat of ethanol (Table 2) but not that of n-propanol (Table 3). Pure n-propanol has a combustion heat of 37 KJ/g, while the blend 3HBME/n-propanol or 3HAME/n-propanol was in the range of 32 to 37 KJ/g, depending on 3HBME content. In addition, the increased contents of 3HBME or 3HAME decreased the combustion heat of the blended fuels from 37 to 32 KJ/g or 35 to 27 KJ/g (Table 3). In the blends of 3HBME/nbutanol and 3HAME/n-butanol, similar trends were observed (Table 3). Cost Estimation of 3HBME or 3HAME. 3HBME or 3HAME production cost can be divided into costs of substrates, fermentation, extraction, methyl esterification, and purification (Table 4 and Figure 2). Because dewatered activated sludge or wastewater can be obtained from wastewater treatment plant without cost, the substrates are considered as free. As such, biomass (activated sludge) or mixed cultures produced from wastewater cost nothing or minimum as sterilization is not needed. For extraction and methyl esterification, current market prices of chemicals including methanol, acetyl butyrate and 98% sulfuric acid are (US$/t) 500, 2000, and 170, respectively. For production of one ton of 3HBME or 3HAME, one ton methanol, 1/4 ton acetyl butyrate, and 1/6 ton sulfuric acid (98%) are consumed. Energy required for gentle heating and mechanical stirring is estimated at about $20/t. In addition, purification
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Table 4. Estimation of the Production Cost (US$/Ton) of 3HBME or 3HAME Obtained from Hydrolysis of PHA Accumulated in Activated Sludge or in Bacteria Grown in Waste Water cost structure
substratea
fermentationb
extraction + esterificationc
purificationd
total coste (per ton)
In US$
0
0-100
1048
50
1098-1198
a
b
Dewatered activated sludge can be obtained from wastewater treatment plant without cost. Biomass (activated sludge) or mixed cultures produced from wastewater cost nothing or a minimum amount, as sterilization is not needed. c Methanol, $500/t; acetyl butyrate, $2000/t; sulfuric acid (98%), $170/t. For one ton of 3HBME or 3HAME, we need one ton methanol, 1/4 ton acetyl butyrate, and 1/6 ton sulfuric acid. Energy required for gentle heating and mechanical stirring is estimated at about $20/t. d Purification includes removing residual water and particles using physical means, including Ca(OH)2, Na2SO4, and so on, and filtration. Unless pure HAM is required, distillation is discouraged, as it is energy intensive. e Compared with US$800/t for gasoline that is still increasing.
Figure 2. Schematic illustration of production of PHA based biofuel 3HBME or 3HAME.
includes removing residual water and particles using physical means including Ca(OH)2, Na2SO4, and so on, and filtration. Unless pure 3HAME is required, distillation is discouraged as it is energy intensive. Combined with all of these factors, we could roughly estimate the cost of one ton 3HBME or 3HAME to be around US$ 1098-1198. Compared with US$800/t in the Chinese market for gasoline, the 3HBME or 3HAME is still more expensive. However, these PHA based biofuels are more sustainable as it is less dependent on exhausting petroleum and food based raw materials. In some mixed cultures or activated sludge, PHA production could be as high as over 50% of the cell dry weight (CDW).29 If pure culture was used, normally over 45% PHA could be produced from some wastewater.30 Under the catalysis of sulfuric acid, the ester bond linkage in the PHA polymers can be cleaved and reacted with methanol
to form the corresponding methyl esters. Major contaminants during 3HBME preparation could be crotonic acid resulted from the β-elimination reaction of PHB.31 In FTIR spectra (Figure 1), the double bond (CdC) of crotonic acid was not observed, suggesting the elimination of crotonic acid from 3HBME after distillation. 3HBME and 3HAME were also distinguishable from the FTIR spectra.32 It was reported that the addition of ethyl alcohol enhanced the combustion heat of gasoline.33 In this study, it was found that 3HBME or 3HAME improved the combustion heat of ethanol with 3HAME having more obvious effects (Table 2). It is important to exploit more new biofuels; also, it is equally important to improve the quality of biofuels such as their combustion heats. With the increased content of 3HBME or 3HAME in the ethanol, the combustion heats of blended fuels remained at the similar levels. This may enhance the possibility
(R)-3-Hydroxyalkanoate Methyl Esters as Biofuels
to use 3HAME as a biofuel because the addition of a low content on 3HAME can have similar effects. Although 3HBME failed to increase the combustion heats of diesel and gasoline, respectively, combustion heats of the blended fuels can still maintain at a relatively high level. It was expected that the addition of 3HAME having a higher combustion heat could improve that of diesel or gasoline. However, combustion heats of 3HAME/diesel or gasoline blended fuels were lower than that of the pure diesel or gasoline. Reasons for this are still unknown, although incomplete burning is a normal phenomenon existing in diesel and gasoline due to the long carbon chain. The 3HAME containing 36 mol % 3HDD methyl ester may also have incomplete burning effect, leading to the reduced combustion heat. The addition of 3HAME may worsen the incomplete burning of diesel or gasoline. Methods including reduction on sample weights, addition of a combustion enhancer, and so on might help tackle this problem. 3HBME or 3HAME significantly enhanced the combustion heat of ethanol but not other alcohol-like fuels including n-propanol and n-butanol (Table 3). Similarly, 3HAME had better effects than that of 3HBME (Table 3). 3HAME can also be used as a fuel alone as it produced a combustion heat of 30 KJ/g, a bit lower than widely accepted biofuel n-propanol and n-butanol. Because PHA production is a common phenomenon among bacteria, close to 30% of the soil bacteria produced PHA.34 At the same time, microbial production of PHA can occur under many circumstances, especially from wastewater. Based on the cost factors of 3HBME or 3HAME production (Table 4), we could calculate the cost of these PHA based biofuels to be around US$1200/ton; this price is still higher than that of the current gasoline products in the Chinese market, which is priced at around US$800/t. However, the increasing petroleum price should make 3HBME or 3HAME more competitive. In addition, PHA production in activated sludge could be promoted by adjusting the C to N ratio in the wastewater,29 such as the blending of volatile fatty acids (VFA) and lipid rich wastewater with nitrogen poor water together to promote the PHA formation. In most cases, the PHA based biofuels will be a mixture of 3HBME or 3HAME. However, this will result in fluctuation of combustion heats. In the case that the 3HBME or 3HAME is only used as an additive to the engines, this should not be a problem.
Conclusion PHA universally synthesized by many bacteria grown in various carbon sources including wastewater can be cleaved and reacted with methanol to form their corresponding methyl esters. The resulting 3HBME and 3HAME improved the combustion heat of ethanol, with 3HAME having more obvious effects. In addition, 3HBME and 3HAME could also be used as fuel additives for other fuels such as propanol, butanol, gasoline, and diesel. This is just the first investigation on the possibility of using PHA as a biofuel. Upcoming research should focus on improving the PHA production in activated sludge or on using wastewater as a fermentation medium to grow high PHA, producing mixed cultures. Because the application of PHA does not require highly purified PHA, the production process appears to be much simpler (Figure 2).
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Acknowledgment. The research was supported by Li KShing Foundation and National High Tech 863 Grant (Project No. 2006AA02Z242 and 2006AA020104), as well as the State Basic Science Foundation 973 (2007CB707804).
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