Recovery of Polyhydroxyalkanoates (PHAs) from Mixed Microbial Cultures by Simple Digestion and Saponification Salmiati1, Z. Ujang 1*, M.R. Salim1 and G. Olsson2** 1
Institute of Environmental and Water Resource Management (IPASA), Universiti Teknologi Malaysia, 81310 Johor, Malaysia. *Email:
[email protected] 2 Lund University, SE-22100 Lund, Sweden, (**Email:
[email protected]) ABSTRACT Highly efficient separation and purification of polyhydroxyalkanoates (PHAs) from PHA-containing cell mass is essential for the production of bioplastics from renewable resources in a cost-effective, environmentally friendly way. Combination of digestion and selective dissolution of non-PHA cell mass (NPCM) using low concentration of sodium hypochlorite of PHA biopolymers, a simple process is developed and has demonstrated to recover PHA from cell mass to high purity (>90 wt %) with high yield (>90 wt %). Saponification was applied to separate or remove oil from the PHA-particles. This method was adopted in this study and was able to increase the PHA extraction from the mixed microbial intracellular biomass up to 85% of cell dried weight (CDW). Keywords: recovery, digestion, PHAs, purification, inclusion body, saponification
INTRODUCTION Polyhydroxyalkanoates (PHAs) are the polyester of hydroxyalkanoates that accumulate as carbon/energy or reducing power storage material in microbial cells. They are synthesized and
stored
by a wide variety of bacteria (both
gram‐positive
and
gram
negative
bacteria)
under stress condition and accumulated as intracellular granules without
hazardous
effects
to
the
hosts
[1‐3]. The
structure,
physio‐chemical
properties,
monomer
composition
and
the
number
and
size
of
the
granules
vary
depending
on
the
organism
and
the carbon source utilized to grow the bacteria [4].
PHA
is
typically
produced
as
a
polymer
of
103
to
104
monomers,
which
accumulate
as
inclusions
of
0.2–0.5
μm
in
diameter.
Separation of particles (0.05-100µm) from biotechnological mixtures of particles, such as inclusion bodies, cell debris, and crystal, is gaining interest from industry because of an increasing number of production processes that yield a particulate product in a mixture with other particles [5]. The extraction and the purification of bacterial polyhydroxyalkanoates are the key step of the process profitability in the fermentation system. The ideal method should lead to a high purity and recovery level at a low production cost. Some improvements have been done to enhance the extraction process. The major step of the separation process is the extraction of PHAs granules. In order to get better recovery, a pretreatment step can be added to the process so the cell disruption is improved. Moreover, a purification step can also be added to the process to get a higher purity. The use of a solvent to recover PHA is one of the oldest methods. The use of solvents destroys the natural morphology of PHA granules that is useful in certain applications such as the production of strong fibers. Another problem connected with the use of solvents is that it creates hazards for the operators and for the environment [6]. Most methods to recover intracellular PHA involve the use of digestion methods. Such a method can reduce the use of large quantities of solvent making the procedure economically and environmentally unattractive [7,8]. Another recovery method is the using of sodium hypochlorite for differential digestion of non-PHA cellular materials (NPCM) [9]. With this method, Hahn et al. [10] obtained high purity levels of PHA: 86% with R. eutropha and 93% with recombinant E. coli.
The purpose of this study was to develop a simpler method using low concentration of the solvent to recover PHA from mixed microbial cultures. Various ratio surfactants were examined for their ability to digest NPCM in mixed microbial cultures for PHA recovery. In more specific, the method is suitable to be used for extracting PHA from biomass containing oil residues. MATERIALS AND METHODS Microorganisms and Culture Conditions PHA was produced by the fed-batch culture of mixed microbial cultures in a defined medium using two double-jacketed laboratory-scale reactor with six liters effective volume. Mixed microbial cellular biomass treating palm oil mill effluent (POME) contains unknown constituents of PHA non-selected bacteria. After fermentation, the culture was harvested and stored at -70oC until required. PHA Recovery by Chloroform Extraction Chloroform and other chlorinated hydrocarbons dissolve all PHA from mixed culture biomass [11]. A method employing both types of solvent (lipid extraction with PHA non-solvent followed by polymer dissolution) is therefore applied in this study. The dissolved polymer is separated from the solvent, usually by evaporation or precipitation with alcohol or acetone, such as methanol. In addition, NPCM by alkaline solutions of sodium hypochlorite was employed to separate this material or remove cell debris (upper and second layer of the separate phase). Even the hypochlorite solution is based on the fact that it can dissolve nearly all components of cell except PHB granules [12,13]. But, it can also attack the PHB granules and causes severe degradation of PHB, rendering the PHB unsuitable for many applications. Although more economical ways of recovering PHB have been improved [12,14], the solvent extraction is widely used to recover PHB with a high purity. There are three steps of the recovery process that are applied in this study: (a) Pre-treatment: When the fermentation process for PHA accumulation has finished, the biomass in 3 L of fermentation was harvested by centrifugation at 3000 rpm (acc ≈ 1000g) for 20 minutes. The residual sludge pellet was washed twice with distilled water and underwent frozen drying at -40oC. (b) Extraction: About 12 gram of biomass powder was treated using 100 ml 6% sodium hypochlorite and 100 ml chloroform. The mixture was agitated in a shaker at 300 rpm at 37oC for 3 hours. After the treatment, the dispersion was centrifuged at 3000 rpm (1000g) for 20 minutes. The three separate phases were obtained. The upper phase was the hypochlorite solution, the middle phase contained NPCM and undisrupted cells, and the bottom phase consisted of the chloroform layer containing PHA. The chloroform layer was obtained by filtration. Separation of PHA-enriched solvent was achieved by using syringe from the bottom part of the centrifuge tube. Then, the PHA material was precipitated by mixing methanol with the concentrated chloroform (methanol: chloroform = 9:1). Finally, the PHA-precipitate was filtered by simple filtration and then dried by evaporation at 60oC. (c) Saponification: After precipitation, the oil is still present in the PHA-evaporate. The PHAevaporate containing both oil and PHA were crushed, and the oil was obtained by a combination of pressing and extraction with solvent that did not dissolve PHA (e.g. hexane). Both oil and PHA were extracted from the PHA-evaporate by crushing in hexane. After removal of hexane, oil, which alone cannot dissolve PHA polymers, becomes an effective suspending medium for the precipitating PHA. After the step of recovering the precipitated PHA from the impure solvent liquor, the solvent liquor can be recovered and recycled and/or reused by traditional means in the processes and methods
herein. The wash solvent filtrate can be recycled directly through precipitation or can be distilled to recover methanol for recycle. Figure 2 shows the steps of the extraction of PHA and recycle of the solvent. Sodium hypochlorite of low (
