PHA Recovery from Biomass - Biomacromolecules (ACS Publications)

Review pubs.acs.org/Biomac

PHA Recovery from Biomass Mohamed H. Madkour,‡ Daniel Heinrich,† Mansour A. Alghamdi,‡ Ibraheem I. Shabbaj,‡ and Alexander Steinbüchel†,‡,* †

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, and ‡Environmental Sciences Department, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah, 21589, Saudi-Arabia ABSTRACT: The recovery of polyhydroxyalkanoates (PHAs) from biomass, that is, from bacterial cells, is one of the major obstacles in the industrial production of these polyesters. Since PHAs are naturally synthesized as intracellular storage compounds for carbon and energy and are for this deposited in the cytoplasm of the bacterial cell, PHAs are more or less tightly linked with the entire biomass, and the polyesters must be released from the cells before their isolation and purification can be conducted. This additional step, that is, the release from the cells, is a major difference from most other biotechnological processes where the product occurs outside of the cells because it is secreted into the medium in a bioreactor or because it is synthesized in vitro in an enzyme reactor in a cell free system. This additional step contributes significantly to the overall costs of production. In this review we provide an overview about the different processes that result in the release of PHA from the cells, and we evaluate these processes with regard to the suitability at large scale in the industry.

■

INTRODUCTION Although oil reserves are decreasing, the demand for plastic is steadily increasing. The global annual plastic production is currently about 300 million tons with an annual increase of about 5%.1 Production of synthetic plastic is, besides fuel, by far the most important use of petroleum, which is currently produced in amounts of about 4 gigatons per annum. With a decrease of available oil, the production of plastic from petrochemical resources will also become limited. Furthermore, synthetic plastic from petrochemical resources is harmful to the environment, since it cannot be completely recycled or because recycling processes are very expensive.2 In addition, the overwhelming fraction of synthetic plastics is persistent with regard to degradation, and since it is more or less nonbiodegradable, it is therefore also not compostable. Furthermore, plastic litter makes the environment to occur unpleasant and provides also harm to animals. The chemical industry is on one side continuing the production of traditional plastics, but on the other side it is meanwhile developing a broad range of biodegradable and compostable plastics. Some of them are produced employing chemical processes, as in the case of Ecoflex by BASF;3 others are produced by a combination of a biotechnological and a chemical process yielding polylactide,4 as is done by the company Natureworks in the U.S.; other such nonpersistant polymers are completely produced by biotechnological processes such as bacterial polyhydroxyalkanoates (PHAs), as shown in Figure 1. The latter is done by several companies, mainly in the Americas and in Asia, at different production scales up to 50.000 tons per year (see ref 5 for a good and recent overview). These “bioplastics” or “green plastics”, which are produced as an alternative to synthetic plastics and which © XXXX American Chemical Society

Figure 1. Generalized chemical structure of polyhydroxyalkanoates: R1/R2, alkyl group; n, 100−30.000; X, 0−4.

are usually degraded by many microorganisms,6,7 gained in importance during the last decades.8 Poly(3-hydroxybutyrate), P(3HB), was described in 1926 as the first member of PHAs when it was discovered as cytoplasmic inclusion bodies in Bacillus megaterium.9 Since the 1960s, PHAs were intensively investigated for their properties and biosynthesis.10 PHAs are thermoplastic polyesters.12 They exhibit mechanical properties similar to those of several synthetic polymers, such as polypropylene, which is obtained from petroleum. PHAs possess obvious ecological advantages over synthetic polymers because they are completely biodegradable and nontoxic.13 Despite the attractive characteristics of PHAs, their use in food packaging, biomedical, and other high-volume applications is still limited to small amounts due to the high production costs.1,14 Due to the facts that PHAs are nontoxic and water-insoluble thermoplastic polymers, a high commercial potential arose from their possible applications in industry, medicine, pharmacy, and agriculture.15 PHAs as bulk cellular constituents are unique to Bacteria and Archaea, and they are intracellularly accumulated in many microorganisms as predominant energy and carbon storage compounds. Accumulation occurs usually if one nutrient, such Received: May 28, 2013

A

dx.doi.org/10.1021/bm4010244 | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Review

Figure 2. Comparison of intra- and extracellular products in biotechnology (A and B). Poly(3HB) accumulating cells of R. eutropha H16 (C): bar, 0.5 μm.

■

INTRACELLULARLY ACCUMULATED PRODUCTS IN BIOTECHNOLOGY The overwhelming fraction of biotechnological products that were successfully established, such as antibiotics, organic acids, technical enzymes, etc., are extracellular products. This is even true for most of the commercial biopolymers which are currently produced, such as xanthan, dextran, schizophylan, cellulose, etc. In contrast and as already mentioned above, PHAs are, like only very few other biotechnological products, intracellularly produced. This has several severe consequences, and it causes limitations regarding not only the fermentative part of the production process but also the downstream processes to obtain the polyesters in a purified state (Figure 2).11 One consequence is the need of high cell density cultivations because the availability of cytoplasmic space limits the amount of PHA that can be produced within the cells. In processes where an extracellular product is formed, or in processes that rely on cell-free enzymatic catalyzes, this limitation does not occur. Therefore, in theory and often also in practice, the amount of a biopolymer produced per volume in a bioreactor by an extracellular process is usually higher than the amount that can be obtained by an intracellular process.11 Another general consequence is that an intracellular product must be in contrast to an extracellular product released from the cells by an additional process step. Furthermore, the release of PHA from the cells will, of course, also release other cell compounds, yielding a less homogeneous starting material for purification. These other compounds must then be separated additionally from the polyester. In the case of PHAs, economically feasible extracellular production processes are not available (see also below), as well as in vitro production processes are also not. Strategies to convert the intracellular PHA production process into an extracellular production

as nitrogen, phosphorus, sulfur, oxygen, or magnesium is limited in the presence of excess carbon source and if growth is thereby imbalanced.2,7,16−19 The chain lengths of 3-hydroxy fatty acids in PHAs range from 3 to 5 carbon atoms in shortchain length PHAs (PHASCL) such as P(3HB)2,11,20 or from 6 to 14 carbon atoms in medium-chain-length PHAs (PHAMCL) such as poly(3-hydroxyoctanoic acid), P(3HO).21−23 In total, about 150 different constituents of PHAs are meanwhile known (Figure 1).24 Ralstonia eutropha H16 produces only PHASCL, such as the homopolyester P(3HB) or the copolyesters poly(3hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), or others. Genetically modified R. eutropha can also incorporate 3-hydroxyhexanoate (3HHx) into the polymer.25 The resulting copolymers, such as P(3HB-co-3HHx), and also P(3HB-co3HV) have better mechanical and thermal properties than the P(3HB) homopolyester.7,26 Apart from the polyesters themselves, the complex granules in which PHA is stored in the cell are also of commercial interest, because they may serve potentially as nanobeads for protein purification or drug delivery systems.27 A last short review on the isolation of bacterial PHAs was written in 2007 and published in the following year.28 In the last five years, new aspects and developments occurred. The current review article deals with one important aspect of PHA production. This is the methodology of PHA isolation from the cells and the recovery of the released PHAs. The advantages and disadvantages of the different methods for recovery and purification will be outlined. Furthermore, this review puts the isolation methods/process in context with the used production organisms as well as with the applicability to large scale. B


Biomacromolecules

Review

Figure 3. Principal methods for PHA recovery from bacterial cells.

process with PHA secretion by metabolic and genetic engineering of the organisms have so far not been successfully applied. There are only very few bacteria known which according to the literature secrete PHAs extracellularly. These are mainly hydrocarbonoclastic bacteria from marine habitats.29 PHA formation by Alkanivorax borkumensis, which is one of these

bacteria, occurs intracellularly, but the polymer is then partially excreted. This is one prominent example.30 In addition to PHAs, lipids are also secreted by this bacterium. However, A. borkumensis itself is not suited for the extracellular production of PHA because the cells grow very slowly and use, besides hydrocarbons, almost no other carbon sources for growth. Unfortunately, the secretion processes for either hydrophobic C


Biomacromolecules

Review

Table 1. PHA Recovery Methods: Advantages and Disadvantages method solvent extraction

disruption by sodium hypochlorite disruption by surfactants disruption by chelatehydrogen peroxide treatment disruption by dissolution of non-PHA cell mass by acids enzymatic cell disruption disruption by bead mill disruption by high pressure homogenization disruption by ultrasonication supercritical fluids cell fragility air classification dissolved-air flotation spontaneous release of PHA granules

advantage

disadvantage

high product purity, no degradation of the polymer, low endotoxin content of PHA extracted from Gramnegative bacteria high product purity, no drying of cells necessary, applicable to large scale, applicable to environmental samples PHA can be recovered directly from the culture broth, limited degradation of the polymer high product purity

with most solvents only applicable to small scale, often dried cells required, economically not feasible, hazardous to human beings and the environment, most suitable for lab scale digestion of the cell matter strongly exothermic, hypochlorite may digest also the polymer, thereby reducing the molecular weight high costs, SDS is difficult to recover and to remove from the isolated polymer

inexpensive and ecologically friendly, high yield and purity of PHA

acids may degrade PHA, leading to a reduced molecular weight

high recovery rate and purity of the polymer, mild operating conditions no chemicals required

high costs for enzymes

good performance at high biomass concentrations, applicable for large scale treatment combination with other extraction methods leads to high purity of the product low cost chemical treatment with moderate operating conditions, nonflammable, low toxicity and reactivity efficient and gentle release of the polymer, high recovery and purity of the product, applicable to several bacteria high purity of the product no chemicals necessary no chemicals necessary

compound are not at all understood,31 and it is therefore not possible to transfer this “PHA secretion system” to other microorganisms. Recovery of PHA usually starts after cell separation from the growth medium at the end of a high cell density cultivation process (Figure 3). The cell density can amount up to 170 g/ L32,33 at large scale. Successful recovery processes of an intracellular biopolymer depend on an efficient cell envelope rupturing and on the subsequent purification process. In addition, a successful process must be applicable at large scale and, in the case of biological processes, it must be also strictly controlled at high cell densities. To simplify the disruption of the cells, pretreatment steps may be applied first to the bacterial biomass.

depending on the conditions, degradation of the polymer may occur with concomitant reduction of molecular weight

requires efficient cooling, several passes necessary for a reasonable recovery, difficult to scale up poor disruption at low biomass concentrations difficult to apply to large scale requires strict process parameters, further chemicals needed for a high degree of disruption genetically engineered production strains required numerous steps to recover the polymer consecutive batch flotation steps required genetically engineered producing strains required, not all cells may secrete PHA granules

Another applicable pretreatment procedure is the use of alkali. It has been reported that pretreatment of cells of Alcaligenes latus with sodium hydroxide reduced the number of passes in a continuous flow bead mill from ten for untreated cells to only three for treated cells to release most of the proteins, which were determined as indicators for the released compounds, at same disruption conditions.37 It turned out that a minimum of 0.12 kg of NaOH per kilogram of biomass was necessary. Another reported pretreatment procedure is the use of a x M sodium chloride salt solution for 1 h at 60 °C with A. latus, as described by Tamer et al.37 Khosravi-Darani et al.38 used 140 mM sodium chloride for cells of R. eutropha. However, nothing is known about applications of these pretreatment methods at large scale in industry. Also, freezing/thawing cycles can be used as a pretreatment or even for the release of the PHA granules from the cells. It was found that such a pretreatment makes the release of PHA by SDS treatment easier.39 The effect is due to the mechanical disruption of the cells by ice crystal formation during freezing.40 However, freezing and thawing of biomass at large scale is very expensive and not feasible. This is probably the reason why this pretreatment has not been reported to be applied in industry. Finally, also the medium composition may have an impact on the accessibility of the cells to cell disruption methods. For example, cells of A.vinelandii grown in fish peptone medium become fragile after having accumulated a large amount of PHA.41 This measure was combined with simple recovery protocols such as treating with 1 N aqueous NH3 (pH 11.4) at 45 °C for 10 min. Cells of A. vinelandii, which were grown in a different medium lacking fish peptone, did not exhibit this feature. Because the selection of an appropriate production medium represents some kind of very indirect and far ahead pretreatment, it is mentioned in the “pretreatment” section of this review. However, these are again measures that must be

■

PRETREATMENT OF CELLS Several pretreatment procedures, which weaken the firmness of the cell wall and envelope and which thereby make the subsequent main biological, chemical, or physical breakage steps which have to be applied to the cells to release the PHA granules from the cells easier and more efficient (Figure 3), were described in the literature. These are mostly processes individually developed for a particular microorganism, and they cannot be applied in a generalized way to all PHA accumulating bacteria because they depend on individual features of the respective bacterial strain. Application of heat is one of these pretreatments that affects the firmness of cells by the denaturation of cell proteins and by the destabilization of the outer membrane.34 Heat pretreatment may also denature the PHB degrading depolymerase enzyme, as was shown for R. eutropha DSM545.34,35 The impacts of variations of temperature and duration were investigated, and 1 min at 120 °C for Pseudomonas cells36 and 15 min at 85 °C for R. eutropha cells35 were reported to be optimal. D


Biomacromolecules

Review

di- or tricarboxylic acid esters such as diethyl succinate, and butyrolactone gave recoveries ranging from 70 to 90% and purities between 99.7 and 100% if high temperatures in the range of 110−140 °C were used.52 Other solvents such as tetrahydrofuran ethyl cyanide, acetic anhydride tetrahydrofuran, or methyl cyanide were also tested but yielded much lower recoveries.53,54 P(3HB-co-3HHx) was efficiently extracted to a reasonable purity from cells of R. eutropha employing several nonhalogenated solvents.56 Acetone is only applicable for extraction of P(3HB) from bacterial cells at high temperatures and high pressures.57 Recently, the use of hot water and ethanol was described for extraction of P(3HB); however, the purity of the isolated polymer was very low (81%).58 Having obtained a solution of the PHA in an organic solvent, the solvent must be removed in a second step. One way is simply via solvent evaporation, leaving the extracted PHA behind in a more or less solvent-free state. However, the inclusion of solvent in the remaining nonevaporatable polymer matrix may be a problem. This can be avoided by aiming at obtaining a thin casted film upon evaporation. This method is therefore only applicable to a small scale and is economically not feasible; however, it may be very useful to isolate small amounts of PHAs, as is often desired in the laboratory. One disadvantage of this method is that non-PHA compounds, which are coextracted by the solvent, will with high probability also remain in the PHA fraction. Another disadvantage of solvent extraction processes is that for most solvents the cells need to be dried first. The latter may be avoided by using a different way to separate the PHA from the solvent. This can be achieved by precipitation of the polymer in the presence of a solvent in which the respective PHA is nonsoluble. These solvents are often referred to as antisolvents. However, for this the PHA solution has to be mixed with a large volume of the nonsolvent, yielding a mixture of at least two different solvents and the precipitated PHA.55 The solution containing the polyester is usually slowly poured into the antisolvent in which the precipitation should occur. The precipitated PHA is then separated from the solvent mixture by centrifugation or filtration. Instead of an organic solvent, also water may be used as the second solvent, as has been successfully demonstrated during the precipitation of P(3HB) from a methylene chloride solution after extraction of the polymer from Alcaligenes latus cells.59 Again, this is a method hardly applicable to industrial large scale under economic premises. Furthermore, recycling of the used solvents will be obligatory if the method is applied on large scale; otherwise, the recovery costs will be too high. New green solvents60 can be applied as well as solubility prediction models for PHAs in various solvents or solvent mixtures.61 In addition, a new process was developed including during solubilization the use of high temperatures close to the melting point temperature of PHA, before the PHA solution was cooled down and pressure was applied.62 A two-phase system composed of polyethylene glycol and phosphate was also successfully applied for the isolation of PHAs. Divyashree et al.63 used this system for the isolation of PHAs from hydrolyzed cells of Bacillus f lexus. Solvent extractions may be too expensive,54 and the separation of polymer may be difficult.6 Another disadvantage of solvent extraction is the harmful effects of solvents on human beings and the environment.6 On the other hand, solvent extraction is probably the most frequent applied method at

individually designed and optimized for one particular microorganism.

■

RELEASE OF PHA FROM CELLS After an eventually applied process to weaken the integrity of the cell wall and membrane layers, biological, chemical, and/or physical processes for the disintegration of the cells to release the polyester molecules or the PHA granules as a whole must be applied. A wide range of methods have been developed in the past and are available (Figure 3). Table 1 summarizes all methods and points to their advantages and disadvantages. 1. Solvent Extraction. The oldest method of PHA recovery is the application of organic solvents to extract the polymer from the cells. In the 1920s Lemoigne9 was the first to describe the isolation of P(3HB) from cells of Bacillus strain M. He achieved extraction of the polyester with hot alcohol (presumably ethanol), and he purified the polyester subsequently with chloroform and diethyl ether. Organic solvents are also applicable to extract P(3HB) from transgenic plants.41 In addition solvents are also applicable to isolate PHAs from environmental samples or sewage sludge, as was successfully done in the laboratories of Wallen42,43 and White.44 Wallen and co-workers were interested to unravel the polymers present in activated sewage sludge from a municipal wastewater treatment plant at Peoria (IL, USA). The studies of this laboratory are of particular historical importance because the results showed already about 40 years ago that, besides P(3HB), also 3hydroxyvalerate, 3-hydroxyhexanoate, and 3-hydroxyheptanoate occur in samples containing biological material. Therefore, he was the first to obtain evidence for the existence of natural polyesters others than P(3HB). He treated a semidry concentrated (by centrifugation) sewage sluge first by a 3fold extraction with hot water. When he then dried the waterextracted sludge with chloroform, he evaporated the chloroform and removed unwanted compounds by treatment with hexane and ether, before he detected all these 3-hydroxyalkanoic acids in the remaining polymer fraction.43 White and co-workers came to similar findings with even a larger number of different 3-hydroxyalkanoates when they extracted lyophilized marine sediments with chloroform.44 Others isolated PHAs from organic waste and analyzed the composition of the extracted polyesters.45 All solvent extraction methods are based on the fact that PHAs are water insoluble but soluble in a limited number of organic solvents. Some chlorinated hydrocarbons such as chloroform, 1,2-dichloroethane, and methylene chloride or some cyclic carbonates such as propylene and ethylene carbonates11,46 and also cyclic carbonic esters47 have been successfully applied as solvents in the past. Furthermore, it was found that these solvents are applicable to most PHAs, in particular to P(3HB) and other PHASCL. More solvents, including acetone, are also applicable to PHAMCL, such as poly(3-hydroxyoctanoic acid).48 PHAMCL are also extractable employing various other nonchlorinated solvents, yielding polyesters with very low endotoxin contents suitable for medical applications.49 Methylene chloride led to purities exceeding 98% of poly(3hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-HV), when isolated from R. eutropha.50 Solvent mixtures, such as chloroform/methanol and dichloromethane/ethanol, were also used.47 Halogenated solvents, such as chloroethanes and chloropropanes, were tested for polymer extraction.51 Alkanediols such as 1,2-propandiol, acetalized triols such as glycerol, E


Biomacromolecules

Review

solvent extraction. Finally, the polymer has to be precipitated in a nonsolvent and filtrated. As shown for cells of R. eutropha containing P(3HB), when applying for 90 min a dispersion of hypochlorite and chloroform consisting of an organic chloroform phase and an aqueous phase with 30% hypochlorite, about 91% of the polyester could be recovered, yielding a material with 97% purity. The procedure decreased the molecular weight of the PHA only marginally, but it requires a large amount of chloroform.69 One advantage of sodium “hypochlorite extraction” is that it can also be applied to environmental samples. This makes the method quite versatile. For example, estauarine detrital microflora, which was used as it was obtained or in the lyophilized state, was treated with a 5% aqueous solution of sodium hypochlorite to recover about 96% of the polyester P(3HB).67 Disruption by Surfactants. Many surfactants, such as sodium dodecyl sulfate (SDS) and others, disintegrate cells by incorporating themself into the lipid bilayer of the cytoplasmic membrane of the cell envelope. This released, for example, P(3HB) from R. eutropha cells into the solution surrounded by the cellular debris66 because the surfactant also solubilized non-PHA cellular materials.72 Lee et al.73 reported on an almost complete lysis of cells of R. eutropha and A. latus in the presence of only 1.0 mM synthetic palmitoyl carnithine within 60 min in 0.1 M Tris−HCl buffer, pH 7.0, at 30 °C. An advantage is that SDS can be used without pretreatments to recover P(3HB) directly from a high cell density culture broth. Using this procedure, a purity of over 95% and a recovery rate of more than 90% of P(3HB) was obtained P(3HB) from R. eutropha cells if a SDS/biomass ratio higher than 0.4 was used.74 To get PHA with a higher recovery and with a purity exceeding 97%, other agents such as hypochlorite and sodium hydroxide must be used in addition.75 A suppressed degradation of the PHAs is the benefit of this method.76,77 However, the costs must be comparably high considering a SDS/biomass ratio on the order of 0.4 and considering also that SDS molecules are, in contrast to organic solvents, not easily removed as well as recovered. Another problem of this method will be the complete removal of remaining SDS from the isolated polymer. Linear alkylbenzene sulfonic acid is superior to SDS because lower amounts per cell mass or PHA are required and because it is environmentally safe.78 A combination of surfactants with chelating agents increased the amount of PHA released from the cells. This method depends on the ability of the chelate to form complexes with divalent cations in the outer membrane of Gram-negative bacteria. Thereby, the outer membrane and subsequently the cytoplasmic membrane are destabilized, and as a result a higher purity of the isolated PHA is obtained. A recovery of 93.3% and a purity of 98.7% were observed under the conditions reported by Chen et al.72 This method yields a large quantity of wastewater. The amount of wastewater can be minimized by recycling the latter for several rounds.79 Disruption by Chelate-Hydrogen Peroxide Treatment. The combined treatment of PHA containing cells with a chelating agent and hydrogen peroxide was used to recover the copolymer P(3HB-co-3HV) with a purity of 99.5% from cells of R. eutropha. This procedure included a pretreatment of the cells for 80 s at 150 °C before the mixture of chelating agent

small scale in the laboratory because it can be applied under standardized conditions to most microorganisms without the need for much optimization. For most other methods described below, optimized protocols have to be established first. Recovery procedures employing organic solvents do not degrade the PHA; that is, the molecular weight and also the molecular weight distribution remain more or less constant. This is very useful, in particular if strong fibers are manufactured.6 Furthermore, the use of solvents reduces the endotoxin content in the PHB extracted from Gram-negative bacteria.64 2. Chemical Disruption. Some chemicals, such as surfactants and sodium hypochlorite, can disrupt the microbial cells, leading to a release of the cell content.65,66 The oxidizing reagent hypochlorite acts because it not only disintegrates the bacterial cell wall but also degrades all other cell constituents by oxidation. Some of these methods are also applicable to collect PHAs from environmental samples.67 Disruption by Sodium Hypochlorite. This method of PHA extraction is based on the treatment of the cells with sodium hypochlorite. This oxidizing agent degrades at appropriate concentrations more or less selectively all polymeric non-PHA cell constituents, whereas PHA mostly resists the chemical attack of hypochlorite and remains. PHA, which does not dissolve in sodium hypochlorite and remains solid, can then easily be separated by filtration or centrifugation.65 One advantage of the hypochlorite method is that the cells need not to be dried before the treatment, although the method is much more effective if dried cells serve as starting material. This saves time and energy and reduces thereby the costs for downstream processing. In general, this process is easy to handle and is with some precautions also applicable to the large scale. Heat formation during the application must be carefully taken care of because the digestion of biomass by hypochlorite is a strongly exothermic process. The temperature in the reaction vessel must be controlled, and an appropriate cooling device must also be used to prevent the formation of foam.68 This was demonstrated in a recent study when PHB was isolated by a simplified method from biomass of R. eutropha.68 The simplifications of the methods consisted of the use of a much higher cell density of 3% (w/v) and the use of only a 13% (w/ v) aqueous solution of sodium hypochlorite. By these measures, the total volume occurring during the application as well as the required amounts of chemicals and energy were significantly reduced. Disruption by sodium hypochlorite can result in a high purity of the polymer with a PHA content in the isolated material of up to 99%.69 The concentration of hypochlorite has to be chosen carefully because PHA is not completely resistant to sodium hypochlorite,65 and a reduction of the molecular weight of the isolated PHA of up to 50% was observed. The hypochlorite extraction method was also applied to isolate high molecular weight poly(3-hydroxypropionate) from cells of a recombinant strain of Shimwellia blattae which was engineered to produce this polyester from glycerol.70 A moderate molecular weight reduction may not cause a problem for many applications because the molecular weight of P(3HB) is usually rather high. Furthermore, the addition of an antioxidant such as sodium bisulfite can diminish this molecular weight reduction.71 The use of a mixture of sodium hypochlorite and chloroform can take advantage of a differential cell lysis by hypochlorite and F


Biomacromolecules

Review

than disruption in a mill. P(3HB) was recovered from cells of Methylobacterium sp. V49 using two cycles in a high pressure homogenizer at a pressure of 400 kg cm−2 and in the presence of 5% (w/v) SDS.91 Disruption by Ultrasonication. Hwang et al.92 studied this method and used it for the isolation of PHA from Haloferax mediterranei. Sonication was also used as a pretreatment before a chemical treatment was applied for various bacteria. Also here, a combination of different methods often gives good results. The combination of homogenization, centrifugation, and sodium hypochlorite treatment led to 96.5% purity and 80% P(3HB) recovery.93 In general, sonication is better applicable at the small laboratory scale than at large industrial scale.94 5. Supercritical Fluids. CO2 as supercritical fluid has a high density and a low viscosity. Therefore, it is a suitable extraction solvent for PHAs. It also has many advantages because it exhibits nonflammability, low toxicity and reactivity, as well as moderate critical temperature and pressure (31 °C and 73 atm), reasonable availability, and low cost. A recovery of 89% P(3HB) from cells of R. eutropha was achieved using supercritical CO2 at an exposure time of 100 min, 200 atm, 40 °C, and 0.2 mL of methanol.95 To enhance the degree of disruption, combinations of supercritical CO2 with NaOH or NaCl for cells of R. eutropha were also successfully used.38 6. Cell Fragility. Recombinant E. coli strains, which were developed for synthesis of P(3HB) at high levels, were additionally established with a lysis system by genetic engineering, in order to provide a novel biological system for cost efficient and easy PHA recovery. The lysis system was based on phage lysis, which allows efficient and gentle release of P(3HB).96 In other laboratories, similar phage lysis systems were also established in recombinant, P(3HB) producing strains of E. coli.97,98 These features were also combined with other measures, such as the addition of 0.2 N NaOH at 30 °C for 1 h or with 5 mL of 1 N sodium hydroxide at 50 °C or with the application of SDS and EDTA or with others,97 and yielded high recoveries and purities of the polyester with high molecular weight. In addition to E. coli, lysis systems for the recovery of P(3HB) were also successfully established in other bacteria. A so-called E-lysis system based on the bacteriophage λ was established in R. eutropha.99 In Bacillus megaterium a selfdisruptive lysis system which is based on the lysis system of B. amyloliquefaciens was successfully established.100 The use of such biological lyses systems for bacterial PHA production is theoretically conclusive and sound and should be able to make a significant contribution to reduce the costs for downstream processing. However, to our best knowledge these systems were never used for the production of PHAs or of other intracellular compounds in industry. This is astonishing because such lysis systems must also be attractive for the production of other compounds, such as, for example, proteins. It is assumed that the strict regulation of the expression and its essential prevention during growth are difficult during high cell density cultivations. 7. Air Classification. PHA granules are released from the cells first by mechanical disruption. The obtained suspension is then dried and converted into a powder by a mill. The next step is air classification to different particle fractions. Subsequently, extraction followed by precipitation yielded the polymer. The application of this procedure to P(3HB) containing cells of R. eutropha led to a fine fraction of 38% and a coarse fraction of 62%. The fine fraction contained the major amount of P(3HB).

and hydrogen peroxide was added. Finally the copolymer was separated by centrifugation.80 Disruption by Dissolution of non-PHA Cell Mass by Acidic and Alkaline Digestion Methods. This is a cheap and green method of PHA recovery with high yield and purity. It decreases the costs of the recovery process by about 90% at large scale production scale, where it is applicable. It includes the selective dissolution of non-PHA cell constituents by protons in an aqueous solution and the crystallization of PHA.81 Similar achievements were made if an alkaline digestion method using NaOH was applied.82 3. Enzymatic Cell Disruption. Some types of enzymes such as proteases, nucleases, lysozyme, and lipases exert high hydrolytic effects on proteins and other polymers of the bacterial cell mass and initiate cell lysis, but they have no or only minor effects on PHA. Already, Merrick and Dourdoroff83 applied lysozyme and deoxyribonucleases for PHA recovery. Holmes and Lim developed a recovery process which included three steps: (i) preheating, (ii) enzymatic treatment, and (iii) surfactant treatment.84 This process was applied to cells of R. eutropha in the industrial production of P(3HB-co-3HV) by Imperial Chemical Industries (ICI) in the U.K.. The use of enzymes gives high recovery but causes also high cost.85 Kapritchkoff et al.34 reported that production of P(3HB) from R. eutropha using pancreatin was three times cheaper than the use of bromelain, and that it yielded P(3HB) with a purity of 90%. Neves and Müller86 compared various commercially available proteases and glucosidases at concentrations of 0.02% (w/w) and obtained purities of more than 93% after 14 h enzyme treatment. Combinations of enzymes and chemicals were also studied. For Burkholderia sp. PTU9 a recovery of 78% and purity of 89% of P(3HB) were reported when using papain with sodium hypochlorite. Using the enzyme alcalase in combination with SDS and EDTA plus a heat pretreatment led to more than 95% medium-chain length PHA recovery from Pseudomonas putida cells. Yasotha et al.87 and Kathiraser et al.88 studied different combinations of alcalase, SDS, and EDTA. They reported 92.6% PHA purity and 90% recovery from the cells after also applying crossflow ultrafiltration and diafiltration. 4. Mechanical Disruption. Mechanical disruption is a commonly used and established method to release proteins from microbial cell mass. These methods are also frequently applied in the laboratory for the release of PHA from bacterial cells on a small scale. However, mechanical disruption methods are less important for industrial large scale PHA production. Disruption by Bead Mill. This method is based on disruption of cells by shear forces caused by solid beads. Scaling up the recovery process is not easy because disruption performance followed a first order disruption kinetics. Efficient cooling must be performed to release the heat formed during mill operation. A complete disruption and reduction of the average diameter of the particles to a few micrometer was achieved after eight passes at 52,800 rpm and 85% loading. The diameter of the grinding beads did not affect the disruption rate in the investigated range.89 Disruption by High Pressure Homogenization. This method of liquid shear disruption is used for large scale biomass treatment.90 The disruption chambers should be kept at an operation temperature of 25 °C. The performance depends on biomass concentration.89 Homogenizers poorly disrupt at low concentrations. At a biomass concentration of 45 kgDWm−3, high pressure homogenization performed better G


Biomacromolecules

Review

released PHA. Depending on the structure and property of the PHA to be isolated, on the production scale, and on the used production strain, different recovery techniques can be selected. Recovery techniques include chemical, enzymatic, and mechanical cell disruption methods as well as solvent extraction and centrifugation and the use of supercritical fluids. The cell’s fragility, air classification, and spontaneous release of PHAs may also be utilized. Further purification may be achieved by hydrogen peroxide or ozone treatments, which have different effects. Biological in vivo as well as in vitro systems for PHA release from the cells to initiate its purification should be paid most attention. These are at first rank (i) phage lyses systems to destroy the cells integrity at the end of the accumulation phase during high cell density cultivation, (ii) the use of technical enzyme cocktails to degrade not only the cell walls but also all other cellular polymers, or (iii) the establishment of an enzymatic in vitro biosynthesis system with efficient cofactor recycling. At second rank these are processes employing (i) hypochlorite treatment or (ii) treatments with hot water or ethanol, which should be preferred against extraction with other organic solvents because the latter cause environmental and cost problems. Such systems should be intensively studied by biologists and engineers because a breakthrough in downstream processing may very likely also result in a breakthrough of the commercial production of PHAs. There are other potential intracellular products that, like PHAs, need the release from the cells and subsequent purification. Besides therapeutic or analytical proteins and technical enzymes, these may be, for example, lipids such as wax esters and triacyl glycerols, which gain recent importance as special chemicals for applications in pharmaceuticals or cosmetics108 and as bulk chemicals for biofuel production.109 Also, the polyamide cyanophycin falls into this category.110 Although these compounds have properties different from those of PHAs, similar principles regarding their production and, in particular, downstream processes apply.

Extraction by chloroform and precipitation by methanol gave P(3HB) with a purity of more than 95% and a yield of about 85%. In the case of E. coli, a purity and a yield of about 97% and 90%, respectively, were achieved.100,101 8. Dissolved-Air Flotation. This method followed an enzymatic disruption step, for example with lysozyme and Novozyme A, to release PHAMCL granules from cells of P. putida. It was found that the isoelectric points of PHA granules and of cell debris were both almost identical at about pH 3.5. After release of the granules, a selective aggregation and then selective flotation controlled by particle−particle interactions, particle−bubble interactions, and hydrodynamics was performed. Using consecutive batch flotation steps, a purity of PHAMCL from P. putida of 86% was obtained.102 9. Spontaneous Release of PHA Granules. Some recombinant strains of E. coli, such as strain MG1655, have been reported to secrete under certain cultivation conditions spontaneously P(3HB) granules from the cells. By centrifugation and washing with distilled water, the phenomenon can be used for purification of P(3HB), as reported by Jung et al.103 Similar is the use of the phaCAB genes from R. eutropha strain H16 in combination with the expression of lysis genes of the E. coli bacteriophage PhiX174 from plasmid pSH2, which led to squeezing of PHB granules out of the cells through the E-lysis channel without any deformation of the cell envelope (see above).104

■

FINAL PURIFICATION OF PHAS Upon the release of PHA granules from the cells and the primary purification step, usually additional efforts must be undertaken, to obtain a sufficiently pure PHA product. There are a large variety of methods available. Which one is finally applied depends very much on the organism from which the PHA was isolated, on the release method, and on the type of PHA on one side. Furthermore, it depends on the final application of the produced PHA. Purification involves frequently treatment with hydrogen peroxide in combination with the enzymatic disruption process or the chelating agents.105 Due to the disadvantages of hydrogen peroxide, which are due to high working temperatures and due to the decrease of the molecular weight of the purified PHA as well as its instability at high concentration in the cellular biomass, this treatment is often replaced by other methods. Ozone treatment is now frequently used to take advantage of its bleaching and deodorization effect and the solubilization of impurities. This treatment increased the ability the remove impurities.106 In the case of large scale polymer production, peptidoglycan is, to a significant extent, blending into the produced polymer latex, thereby affecting the recovery process. Exposing such a PHA latex to an additional solvent extraction led to higher purity. For this, chloroform is preferred because of its lower consumption.107

■

AUTHOR INFORMATION

Corresponding Author

*Mailing address: Alexander Steinbüchel Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische WilhelmsUniversität Münster, Corrensstraße 3, D-48149 Münster, Germany. Phone: 49-251-8339821. Fax: 49-251-8338388. Email: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was funded by the King Abdulaziz University (Jeddah, Saudi Arabia) under grant No. (2- 10- 1432/HiCi). Therefore, the authors acknowledge the technical and financial support of KAU.

■

■

CONCLUSIONS AND PERSPECTIVES Recovery of PHAs from the produced biomass is a very important part of the entire polymer production process. Since PHAs are intracellular products, the downstream processes may be tedious and may contribute considerably to the total production costs. PHA recovery not only requires the release of the polyester from the cells by various means but also includes pretreatment of the biomass and purification steps of the

REFERENCES

(1) Siracusa, V.; Rocculi, P.; Romani, S.; Rosa, M. D. Trends Food Sci. Technol. 2008, 19, 634−643. (2) Doi, Y. Microbial polyesters, 1st ed.; VCH: New York, 1990. (3) Witt, U.; Einig, T.; Yamamoto, M.; Kleeberg, I.; Deckwer, W.-D.; Müller, R. J. Chemosphere 2001, 44, 289−299. (4) Ahmed, J.; Varshney, S. K. Int. J. Food Prop. 2011, 14, 37−58. (5) Chen, G.-Q. Chem. Soc. Rev. 2009, 38, 2434−2446.

H


Biomacromolecules

Review

(6) Byrom, D. Polyhydroxyalkanoates. In Plastics from Microbes: Microbial Synthesis of Polymers and Polymer Precursors; Mobley, D. P., Ed.; Hanser: Munich, 1994; pp 5 ff. (7) Anderson, A. J.; Dawes, E. A. Microbiol. Rev. 1990, 54, 450−472. (8) Sudesh, K.; Iwata, T. Clean-Soil Air Water 2008, 36, 433−442. (9) Lemoigne, M. Bull Soc. Chim. Biol (Paris) 1926, 8, 770−782. (10) Lu, J.; Tappel, R. C.; Nomura, C. T. J. Macromol. Sci., Part C.: Polym. Rev. 2009, 49, 226−48. (11) Steinbüchel, A. Macromol. Biosci. 2001, 1, 1−24. (12) Baptist, J. N. U.S. Patent 3,044,942, 1962. (13) Gogolewski, S.; Jovanovic, M.; Perren, S. M.; Dillon, J. G.; Hughes, M. K. J. Biomed. Mater. Res. 1993, 27, 1135−1148. (14) Steinbüchel, A. In Biotechnology, 2nd ed.; Rehm, H. J., Reed, G., Pühler, A., Stadler, P., Eds.; Wiley/VCH: Weinheim, 1996; pp 403 ff. (15) Lee, S. Y.; Choi, J.; Wong, H. H. Int. J. Biol. Macromol. 1999, 25, 31−36. (16) Lee, S. Y. Biotechnol. Bioeng. 1996, 49 (1), 1−14. (17) Shang, L.; Jiang, M.; Chang, H. N. Biotechnol. Lett. 2003, 25, 1415−1419. (18) Steinbüchel, A.; Füchtenbusch, B. Trends Biotechnol. 1998, 16, 419−427. (19) Verlinden, R. A. J.; Hill, D. J.; Kenward, M. A.; Williams, C. D.; Radecka, I. J. Appl. Microbiol. 2007, 102, 1437−1449. (20) Kunioka, M.; Nakamura, Y.; Doi, Y. Polym. Commun. 1998, 29, 174−176. (21) De Smet, M. J.; Eggink, G.; Witholt, B.; Kingma, J.; Wynberg, H. J. Bacteriol. 1983, 154, 870−878. (22) Eggink, G.; de Waard, P.; Huijberts, G. N. M. FEMS Microbiol. Rev. 1992, 103, 159−164. (23) Timm, A.; Steinbüchel, A. Appl. Environ. Microbiol. 1990, 56, 3360−3367. (24) Steinbüchel, A.; Valentin, H. FEMS Microbiol. Lett. 1995, 128, 219−228. (25) Budde, C. F.; Riedel, S. L.; Willis, L. B.; Rha, C.; Sinskey, A. J. Appl. Environ. Microbiol. 2011, 77, 2847−2854. (26) Gorenflo, V.; Schmack, G.; Vogel, R.; Steinbüchel, A. Biomacromolecules 2001, 2, 45−57. (27) Grage, K. Biomacromolecules 2009, 10, 660−669. (28) Jacquel, N.; Lo, C.-W.; Wei, Y.-H.; Wu, H.-S.; Wang, S. S. Biochem. Eng. J. 2008, 39, 15−27. (29) Manilla-Perez, E.; Lange, A. E.; Luftmann, H.; Robeneck, H.; Steinbüchel, A. Eur. J. Lipid Sci. Technol. 2011, 113, 8−17. (30) Sabirova, J. S.; Ferrer, M.; Lünsdorf, H.; Wray, V.; Kalscheuer, R.; Steinbüchel, A.; Timmis, K. N.; Golyshin, P. N. J. Bacteriol. 2006, 188, 8452−8459. (31) Manilla-Perez, E.; Reers, C.; Baumgart, M.; Hetzler, S.; Reichelt, R.; Malkus, U.; Kalscheuer, R.; Wältermann, M.; Steinbüchel, A. J. Bacteriol. 2010, 192, 643−656. (32) Riesenberg, D.; Guthke, R. Appl. Microbiol. Biotechnol. 1999, 51, 422−430. (33) Lee, S. Y. Biotechnol. Bioeng. 1996, 49, 1−14. (34) Kapritchkoff, F. M.; Viotti, A. P.; Alli, R. C. P.; Zuccolo, M.; Pradella, J. G. C.; Maiorano, A. E.; Miranda, E. A.; Bonomi, A. J. Biotechnol. 2006, 122, 453−462. (35) Saegusa, H.; Shiraki, M.; Kanai, C.; Saito, T. J. Bacteriol. 2001, 183, 94−100. (36) De Koning, G. J. M.; Witholt, B. Bioprocess Eng. 1997, 17, 7−13. (37) Tamer, I. M.; Moo-Young, M.; Chisti, Y. Bioprocess Eng. 1998, 19, 459−468. (38) Khosravi-Darani, K.; Vasheghani-Farahani, E.; Shojaosadati, S. A.; Yamini, Y. Biotechnol. Prog. 2004, 20, 1757−1765. (39) Dong, Z.; Sun, X. Chin. Sci. Bull. 2000, 45, 252−255. (40) Karlsson, J. O. M.; Cravalho, E. G.; Toner, M. Cryo Lett. 1993, 14, 323−335. (41) Murthy, G. S.; Kumar, D.; Strauss, S.; Dalton, D.; Vionocur, J. Am. Soc. Agric. Biol. Eng. Annu. Int. Meet. 2010, 6, 4461−4471. (42) Wallen, L. L.; Davis, E. N. Environ. Sci. Technol. 1972, 6, 161− 164.

(43) Wallen, L. L.; Rohwedder, W. K. Environ. Sci. Technol. 1974, 8, 576−579. (44) Findlay, R. H.; White, D. C. Appl. Environ. Microbiol. 1983, 45, 71−78. (45) Linton, E.; Rahman, A.; Viamajala, S.; Sims, R. C.; Miller, C. D. Water Sci. Technol. 2012, 66, 1000−1006. (46) Fiorese, M. L.; Freitas, F.; Pais, J.; Ramos, A. M.; de Aragao, G. M. F.; Reis, M. A. M. Eng. Life. Sci. 2009, 6, 454−461. (47) Lafferty, R. M.; Heinzle, E. U.S. Patent 4,101,533, 1978. (48) Elbahloul, A.; Steinbüchel, A. Appl. Environ. Microbiol. 2009, 75, 643−651. (49) Wampfler, B.; Ramsauer, T.; Rezzonico, S.; Hischier, R.; Köhling, R.; Thöny-Meyer, L.; Zinn, M. Biomacomolecules 2010, 11, 2716−2723. (50) Zinn, M.; Weilenmann, H. U.; Hany, R.; Schmid, M.; Egli, T. H. Acta. Biotechnol. 2003, 23, 309−316. (51) Vanlautem, N.; Gilain, J. U.S. Patent 4,310,684, 1982. (52) Traussnig, H.; Kloimstein, E.; Kroath, H.; Estermann, R. U.S. Patent 4,968,611, 1990. (53) Matsushita, H.; Yoshida, S.; Tawara, T. Japanese Patent 07,79,788, 1995. (54) Schmidt, J.; Biederman, B.; Schmiechen, H. D. E. German Patent DE 223,428, 1985. (55) Hocking, P. J.; Marchessault, R. H. In Chemistry and Technology of Biodegradable Polymers; Griffin, G. J. L., Ed.; Blackie: London, 1994; pp 48−96. (56) Riedel, S. L.; Brigham, C. J.; Budde, C. F.; Bader, J.; Rha, C.; Stahl, U.; Sinskey, A. J. Biotechnol. Bioeng. 2013, 110, 461−470. (57) Koller, M.; Bona, R.; Chiellini, E.; Braunegg, G. Biotechnol. Lett. 2013, 35, 1023−1028. (58) Mohammadi, M.; Hassan, M. A.; Phang, L.-Y.; Ariffin, H.; Shirai, Y.; Ando, Y. Biotechnol. Lett. 2011, 34, 253−259. (59) Hänggi, U. J. In Novel Biodegradable Microbial Polymers; Dawes, E. A., Ed.; Kluwer: Dordrecht, 1990; pp 65−70. (60) Clark, J. H.; Deswarte, F. E. I.; Farmer, T. J. Biofuels, Bioprod. Biorefining 2009, 3, 72−90. (61) Binder, K.; Müller, M.; Virnau, P.; MacDowell, L. G. Adv. Comput. Simul. Approach Soft Matter Sci. 2005, 173, 1−104. (62) Terada, M.; Marchessault, R. H. Int. J. Biol. Macromol. 1999, 25, 207−215. (63) Divyashree, M. S.; Shamala, T. R.; Rastogi, N. K. Biotechnol. Bioproc. Eng. 2009, 14, 482−489. (64) Lee, S. Y.; Choi, J. I.; Han, K.; Song, J. Y. Appl. Environ. Microbiol. 1999, 65, 2762−2764. (65) Berger, E.; Ramsay, B. A.; Ramsay, J. A.; Chavarie, C.; Branuegg, G. Biotechnol. Tech. 1989, 3, 227−232. (66) Ramsay, J. A.; Berger, E.; Ramsay, B. A.; Chavarie, C. Biotechnol. Tech. 1990, 4, 221−226. (67) Heron, J. S.; King, J. D.; White, D. C. Appl. Environ. Microbiol. 1978, 35, 251−257. (68) Heinrich, D.; Madkour, M. H.; Al-Ghamdi, M. A.; Shabbaje, I. I.; Steinbüchel, A. AMB Express 2012, 2, 60. (69) Hahn, S. K.; Chang, Y. K.; Kim, B. S.; Chang, H. N. Biotechnol. Bioeng. 1994, 44, 256−261. (70) Heinrich, D.; Andreeßen, B.; Madkour, M. H.; Al-Ghamdi, M. A.; Shabbaj, I. I.; Steinbüchel, A. Appl. Environ. Microbiol. 2013, 79, 3582−3589. (71) Roh, K. S.; Yeom, S. H.; Yoo, Y. J. Biotechnol. Technol. 1995, 9, 709−712. (72) Chen, Y.; Chen, J.; Yu, C.; Du, G.; Lun, S. Process Biochem. 1999, 34, 153−157. (73) Lee, K. M.; Chang, H. N.; Chang, Y. K.; Kim, B. S.; Hahn, S. K. Biotechnol. Tech. 1993, 7, 295−300. (74) Kim, M.; Cho, K. S.; Ryu, H. W.; Lee, E. G.; Chang, Y. K. Biotechnol. Lett. 2003, 25, 55−59. (75) Jacquel, N.; Lob, C. W.; Yu-Hong Wei, Y. H.; Wu, H. S.; Wang, S. S. Biochem. Eng. J. 2008, 39, 15−27. (76) Choi, J.; Lee, S. Y. Appl. Microbiol. Biotechnol. 1999, 51, 13−21. (77) Choi, J. I.; Lee, S. Y. Biotechnol. Bioeng. 1999, 62, 546−552. I


Biomacromolecules

Review

(78) Yang, Y.-H.; Brigham, C.; Willis, L.; Rha, C.; Sinskey, A. Biotechnol. Lett. 2011, 33, 937−942. (79) Chen, Y.; Yang, H.; Zhou, Q.; Chen, J.; Gu, G. Process Biochem. 2001, 36, 501−506. (80) Liddell, J. M.; Locke, T. J. U.S. Patent 5,691,174, 1997. (81) Yu, J.; Chen, L. X. L. Biotechnol. Prog. 2006, 22, 547−553. (82) Anis, S. N. S.; Nurhezreen, M. I.; Sudesh, K.; Amirul, A. A. Appl. Biochem. Biotechnol. 2012, 167, 524−535. (83) Merrick, J. M.; Doudoroff, M. J. Bacteriol. 1964, 88, 60−71. (84) Holmes, P. A.; Lim, G. B. U.S. Patent 4,910, 145, 1990. (85) Harrison, S. T. Biotechol. Adv. 1991, 9, 217−240. (86) Neves, A.; Müller, J. Biotechnol. Prog. 2012, 28, 1575−1580. (87) Yasotha, K.; Aroua, M. K.; Ramachandran, K. B.; Tan, I. K. P. Biochem. Eng. J. 2006, 30, 260−268. (88) Kathiraser, Y.; Aroua, M. K.; Ramachandran, K. B.; Tan, I. K. P. J. Chem. Technol. Biotechnol. 2007, 82, 847−855. (89) Tamer, M.; Moo-Young, M.; Chisti, Y. Ind. Eng. Chem. Res. 1998, 37, 1807−1814. (90) Harrison, S. T.; Chase, H. A.; Dennis, J. S. Bioseparation 1991, 2, 155−166. (91) Ghatnekar, M. S.; Pai, J. S.; Ganesh, M. J. Chem. Technol. Biotechnol. 2002, 77, 444−449. (92) Hwang, K. J.; You, S. F.; Don, T. M. J. Chin. Inst. Chem. Eng. 2006, 37, 209−216. (93) Van Wegen, R. J.; Ling, Y.; Middelberg, A. P. J. Chem. Eng. Res. Des. 1998, 76, 417−426. (94) Harrison, S. T. L. Biotechnol. Adv. 1991, 9, 217−240. (95) Hejazi, P.; Vasheghani-Farahani, E.; Yamini, Y. Biotechnol. Prog. 2003, 19, 1519−1523. (96) Fidler, S.; Dennis, D. FEMS Microbiol. Rev. 1992, 103, 231−235. (97) Yin, J.; Xu, Y.; Yu, H. M.; Zhou, P. J.; Shen, Z. Y. Prog. Biotechnol. 2000, 16, 213−218. (98) Yu, H. M.; Shi, A.; Yin, J.; Shen, Z. Y.; Yang, S. L. J. Chem. Technol. Biotechnol. 2003, 78, 283−286. (99) Schroll, G.; Resch, S.; Gruber, K.; Wanner, G.; Lubitz, W. J. Biotechnol. 1998, 66, 211−217. (100) Noda, I. U.S. Patent 5,849,854, 1998. (101) Noda, I. U.S. Patent 5,821,299, 1998. (102) Van Hee, P.; Elumbaring, C. M. R. A.; van der Lans, R. G. J. M.; van der Wielen, L. A. M. J. Colloid Interface Sci. 2006, 297, 595− 606. (103) Jung, I. L.; Phyo, K. H.; Kim, K. C.; Park, H. K.; Kim, I. G. Res. Microbiol. 2005, 156, 865−873. (104) Resch, S.; Gruber, K.; Wanner, G.; Slater, S.; Dennis, D.; Lubitz, W. J. Biotechnol. 1998, 65, 173−182. (105) Suzuki, D. V.; Carter, J. M.; Rodrigues, M. F. A.; da Silva, E. S.; Maiorano, A. E. World J. Microbiol. Biotechnol. 2008, 24, 771−775. (106) Horowitz, D.; Brennan, E. U.S. Patent 20,010,006,802, 2001. (107) DeKoning, G. J. M.; Kellerhals, M.; van Meurs, C.; Witholt, B. Bioprocess Eng. 1997, 17, 15−21. (108) Thomson, N.; Summers, D.; Sivaniah, E. Soft Matter 2010, 6, 4045−4057. (109) Röttig, A.; Wenning, L.; Bröker, D.; Steinbüchel, A. Appl. Microbiol. Biotechnol. 2010, 85, 1713−1733. (110) Mooibroek, H.; Oosterhuis, N.; Giuseppin, M.; Toonen, M.; Franssen, H.; Scott, E.; Sanders, J.; Steinbüchel, A. Appl. Microbiol. Biotechnol. 2007, 77, 257−267.

J


PHA Recovery from Biomass - Biomacromolecules (ACS Publications)

Recommend Documents