Utilization of vinasse for production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) by Haloferax mediterranei

Polyhydroxyalkanoates (PHAs) are natural, renewable and biocompatible biopolymers which can be made into plastic materials with properties similar to petrochemical plastics. Produced intracellularly in bacteria, PHAs can replace synthetic plastics in many applications. However, high production cost of the biopolymers and concurrent availability of low-cost petrochemical-derived plastics make PHA manufacture economically unattractive. To make the PHA production financially more appealing, the use of inexpensive substrates has been the subject of many investigations (Verlinden et al. [2011]).

The effluents from molasses based distilleries contain large amounts of dark brown colored molasses spent wash or vinasse. In the distillation process, ethanol ranges from 5% to 12% by volume, hence it follows that the amount of waste varies from 88% to 95% by volume of the alcohol distilled. An average molasses based distillery generates 15 l of vinasse per liter of alcohol produced. Vinasse is one of the most difficult waste products to dispose because of low pH, high temperature, dark brown color, high ash content and high percentage of dissolved organic and inorganic matter (Pant and Adholeya, [2007]). In attempts to utilize vinasse as a low cost carbon source for commercial synthesis of PHAs, polyhydroxybutyrate (PHB) was produced from spent wash obtained from a distillery producing ethanol by fermentation of sugarcane molasses (Khardenavis et al. [2009]) as well as rice grain and jowar grain based distillery vinasse (Khardenavis et al. [2007]) by mixed culture of microbes.

Halophilic microorganisms are an important source of PHAs and hold promise for providing an economically competitive industrial scale production process. Haloarchaeal strains employed as PHA producers have many advantages. As there are very few microorganisms that are able to survive and grow at high salinities, the risks of microbial contamination can be reduced. Haloarchaea can be easily lysed in distilled water, therefore, the use of large quantities of organic solvents can be avoided and time for PHA preparation can be saved. Cheap carbon sources can be used to synthesize PHAs by haloarchaea and so the cost of PHA production will be lowered (Quillaguamán et al. [2010]).

In our previous article (Pramanik et al. [2012]), we provided the first report of PHB production by a halophilic microorganism, Haloarcula marismortui utilizing vinasse. Our long term objective is to expand the possibility of PHA production by other extreme halophiles consuming vinasse. Thus the specific questions to be answered in this study were: can the methods developed be applied for the production of biopolymers other than PHB? Is the product obtained of high purity? Is reasonable yield and productivity achieved? Although the family Halobacteriaceae includes 30 genera, currently, only a few haloarchaeal strains belonging to the genera Haloferax, Haloarcula, Haloquadratum, Haloterrigena, Halorhabdus, Halobiforma and Halopiger are found to accumulate PHAs. Haloferax mediterranei is so far the best PHA producer of the family Halobacteriaceae. The genes, molecular basis and functional genomics of PHA synthesis by H. mediterranei and related archaea were studied by Xiang’s research group (Liu et al. [2011] and relevant references therein). Therefore, it was our objective to cultivate H. mediterranei in vinasse, isolate the polymer and study its properties.

Due to its high growth rate, metabolic versatility and genetic stability H. mediterranei has become an interesting microorganism for investigating PHA production. The PHA accumulated by H. mediterranei (PHBV), has much better mechanical properties than PHB, (the most frequently occurring PHA) and hence is more promising for commercial production and application. Furthermore, H. mediterranei can accrue PHBV up to 60% (wt/wt) from starch, glucose or other cheaper industrial by-products without the addition of costly and cellular toxic carbons, such as propionic acid or valeric acid that are generally required by producing eubacteria as precursors of the 3-hydroxyvalerate (HV) unit. Therefore, H. mediterranei has become one of the most potential candidate organisms for industrial PHA production (Lu et al. [2008]).

Vinasse is extremely polluting due its high organic load and antibacterial activity which arises from the presence of phenolic compounds (Martín Santos et al. [2003]) that inhibit the PHA producing microorganism, H. mediterranei. Thus, pre-treatment for removal of polyphenolic compounds was necessary and the process proved to be effective as 25-50% of an acutely polluting waste like vinasse could be utilized. [Gurieff and Lant (2007]) noted that an effective industrial wastewater treatment process concurrently associated with PHA production would always be a desirable technology. Koller et al. ([2007]) observed that in Europe utilization of regionally arising waste and surplus materials, such as whey for the production of PHA should be forcefully aspired.

Significant improvements in the bioprocessing aspects compared to our previous study (Pramanik et al. [2012]) were achieved in this investigation. The two drawbacks in our earlier report were, first, the lag phases of growth of Haloarcula marismortui were prolonged and second, 85% organic carbon remained unutilized in cultures containing 100% pre-treated vinasse. The first limitation was overcome in the present study by using a concentrated inoculum and the second shortcoming was not observed in the present study. Perhaps high growth rate, metabolic versatility and genetic stability of H. mediterranei allowed complete utilization of 25% and 50% of pre-treated vinasse. The reduction of BOD₅ and COD of the pre-treated vinasse were 23% and 35% respectively (Pramanik et al. [2012]), while at least 78% and 80% lowering of BOD₅ and COD were achieved in this study. Comparing the kinetic parameters, five to six fold improvement in the specific production rate and ten fold rise in the volumetric productivity have been attained in the present study. Thus, H. mediterranei demonstrated more potential than H. marismortui for further development to higher scales.

H. mediterranei accumulated 50 wt.-% of poly-3-(hydroxybutyrate-co-8%-hydroxyvalerate) and 73 wt.-% of poly-3-(hydroxybutyrate-co-6%-hydroxyvalerate) from hydrolyzed whey permeate (Koller et al. [2007]; Koller et al. [2008]). In another study, Huang et al. ([2006]) reported 55.6 wt.% accumulation of PHA by the haloarcheon employing extruded rice bran and extruded corn starch. Similarly, Chen et al. ([2006]) applied native corn starch treated by an enzymatic reactive extrusion process leading to 50.8% (w/w) dry cell PHBV content (10.4 mol % HV) in H. mediterranei. The present investigation is the first report on the accumulation of 70% of PHBV (12.36 and 14.09 mol % HV depending on the substrate) by H. mediterranei utilizing vinasse. Recently the composition of vinasses has been published by Parnaudeau et al. ([2008]). Sugarcane vinasse contains significant amounts of sucrose as well as oxalate, lactate, malate and pyruvate which are ready metabolites to be fed to the tricarboxylic acid cycle. Acetate, valerate, propionate and butyrate may have provided the precursors for HV biosynthesis. The described process may be less expensive than the whey process where costly ultrafiltration step is required. After removing 80% of water (which is also energy intensive) from the sweet skimmed whey, the concentrate is ultra filtrated to obtain the whey permeate (Koller et al. [2007]). Further, an additional pre-treatment step for lactose hydrolysis (enzymatically or chemically) would enhance the cost of production. In the processes employing rice bran and corn starch (Chen et al. [2006]; Huang et al. [2006]), the nutrients of these methods would incur a raw material cost. The outlay would further increase as expensive extrusion machinery and α-amylase will be required. Therefore, among the currently described processes for production of PHA by H. mediterranei employing cheap carbon sources, the described method utilizing vinasse is the most economical. Precursors of 3-hydroxyvalerate are known to enhance the 3-HV content in Haloferax mediterranei. Starting from the 3-HV content (6.0%) formed by H. mediterranei from hexoses of whey sugars as sole carbon source, Koller et al. ([2007]) triggered the content of 3-HV to 22% by addition of 1.0 g/l sodium valerate, a 3-HV precursor. However, the cosubstrates are expensive and add to the production cost as estimated by [Choi and Lee (1999]). Our objective was to utilize a waste product and the effect of many nutrients on 3-HV content was not considered to be studied. Increasing concentration of the vinasse enhances the 3-HV content marginally, probably due to the higher amounts of organic acids available. Huang et al. ([2006]) and Chen et al. ([2006]) did not examine the effect of other additional nutrients on the content of 3-HV.

Salts concentration in the culture medium used for cultivation of H. mediterranei should be maintained above 22% (w/v) for optimum cell growth and PHA production (Quillaguamán et al. [2010]). At such a high concentration of salt, the growth of non-halophilic microorganisms is prevented hence allowing a process without strict sterile conditions. The costs of energy required for sterilization can be avoided. Reduction in costs of process piping, instrumentation and insulation as well as saving on electricity for steam generation can be envisaged. A halophilic bacterium, Halomonas TD01 isolated from a salt lake in Xinjiang, China was cultivated in an unsterile and continuous fermentation process (Tan et al. [2011]) that opened a new area for reducing the cost in PHA production. The authors produced PHB, the most common PHA while we obtained PHBV that has better mechanical properties than PHB. Tan et al. ([2011]) applied a designed synthetic medium in their unsterile operation while we used waste spent wash direct from the final ethanol distillation column in our unsterile cultivation which is more prone to contamination. We have obtained a product as pure as that marketed by Sigma-Aldrich through a simple isolation process. The isolation methods, purity and molecular characterization of the polymer were not detailed by Tan et al. ([2011]). We carried out an intensive molecular analyses of the polymer obtained through an alternative zero cost carbon source before embarking on a large scale production system which is planned for the future.

The major step of the separation process is the extraction of PHA granules (Jacquel et al. [2008]). Extraction using solvents such as chloroform, methylene chloride, propylene carbonate and dichloroethane resulted in very pure PHA (Ramsay et al. [1994]). This extraction method required large quantities of toxic and volatile solvent, which not only increased the total production cost but also had adverse environmental consequences ([Choi and Lee 1997]). During digestion of non-PHA cellular materials using hypochlorite, severe degradation of PHA was observed. An enzymatic digestion method developed by Zeneca was used for the production of Biopol (PHBV) but the use of expensive chemicals and complex processes did not seem to be economical ([Holmes and Lim 1990]). The extraction step of the described procedure consumed no energy and lesser quantities of chemicals compared to other recovery methods such as solvent and supercritical fluid extraction, digestion by sodium hypochlorite and enzymes as well as mechanical disruption of cell walls by high-pressure homogenization and ultrasonication (Jacquel et al. [2008]). As the PHA granules themselves do not contain much contaminant (Steinbüchel et al. [1995]), highly pure PHA can be produced by processes that break the cell and solubilize cellular material other than PHA. Therefore, a simple digestion method by inexpensive chemicals seems to be the most efficient and economical recovery process. Subsequent purification steps were essential to obtain a high-purity product. Surfactant pre-treatment and hypochlorite digestion under optimized conditions resulted in a very pure PHB with less degradation (Choi and Lee, [1999]) and was applied for post-extraction purification in the present study.

Nonato et al. ([2001]) observed that PHB and related copolymers can be advantageously produced when integrated into a sugarcane mill. This model made extensive use of facilities, materials and surplus energy from the sugarcane industry that would otherwise be wasted or sold at subsistence prices. We believe that amalgamation of PHA production in an ethanol manufacturing unit, as projected in this study is superior to integration in a sugar mill because the PHB production cost in the sugar mill was heavily dependent on sugar prices and accounted for nearly 29% of the final cost. Such concerns should not arise when a highly polluting waste as vinasse is utilized and the process integrated within ethanol manufacture.

The price of production of 1 kg poly-3-(HB-co-HV) was calculated to be € 2.82 at a volumetric productivity of 0.29 g/l h when using hydrolyzed whey as carbon source for H. mediterranei. This price is significantly lower than that calculated for the production of PHA by recombinant E. coli at ~ € 4.0 (Quillaguamán et al. [2010]). High PHA content, productivity, zero-cost carbon source, low-cost of isolation of a high-purity product and potential for integration into ethanol manufacturing unit would merit further development of this process to higher scales.