Patent Application: US-201414316363-A

Abstract:
a method for producing polyhydroxyalkanoate comprises the steps of culturing in a culture medium comprising pyrolysis wax obtained from the pyrolysis of polyethylene and optionally a surfactant one or more bacterial strains which are capable of accumulating pha from pyrolysis wax , and recovering the pha from the culture medium . typically , the bacterial strains are selected from the group consisting of : a . calcoaceticus bd413 ; a . calcoaceticus rr8 ; b . sepacia rr10 ; p . aeruginosa 3924 ; p . aeruginosa gl - 1 ; p . aeruginosa pao1 ; p . aeruginosa rr1 ; and p . olevorans .

Description:
broadly , the invention relates to a method for converting non - biodegradable polyethylene to biodegradable polyhydroxyalkanoate . the method involves performing pyrolysis on the pe to generate a liquid biodiesel fraction and a waxy by - product known as pe pyrolysis wax . the pe pyrolysis wax is then used as a substrate in a bacterial culture medium comprising certain strains of bacteria capable of metabolising the pe pyrolysis wax and accumulating pha . the bacteria capable of metabolising pe pyrolysis wax include a . calcoaceticus bd413 ; a . calcoaceticus rr8 ; b . sepacia rr10 ; p . aeruginosa 3924 ; p . aeruginosa gl - 1 ; p . aeruginosa pao1 ; p . aeruginosa rr1 ; and p . olevorans . two of these strain are capable of growth and accumulation of pha using pe pyrolysis wax as the sole energy source . for the remaining strains , a surfactant , preferably a rhamnolipid , must be added to the culture broth to achieve both bacterial growth and pha accumulation . in this specification , the term “ polyhydroxyalkanoate ” or “ pha ” are used interchangeably and refer to a range of biodegradable polymers that consist of polyesters of ( r )- 3 - hydroxyalkanoic acids . in this specification , the term “ polyethylene ” or “ pe ” are used interchangeably and refer to a non - biodegradable thermoplastic polymer generally having the chemical formula ( c 2 — h 4 ) n h 2 . in this specification , the term “ pyrolysis ” refers to the process of thermochemical decomposition of matter in the absence of oxygen . it is generally achieved by heating in the absence of oxygen to a temperature of at least 300 ° c ., and often much higher , resulting in the long chain hydrocarbons being converted to shorter chain hydrocarbons , and a great increase in the elemental carbon content of the matter . specific methods of performing pyrolysis on pe to produce biodiesel and pe pyrolysis wax are described in ( conesa , et al ., 1994 , wallis & amp ; bhatia , 2007 , aguado , et al ., 2009 , rasul jan , et al ., 2010 , kumar , et al ., 2011 ). the term “ surfactant ” refers to an organic compound that is amphiphilic , having both a hydrophilic domain and a hydrophobic domain . in particular , the term refers to a biosurfactant which is a surfactant produced by a living cell , particularly microbial cells such as bacteria . most biosurfactants are glycolipids , comprising a carbohydrate attached to a long aliphatic side chain , whereas others like lipopeptides and lipoproteins are more complex . examples of biosurfactants include rhamnolipids ( produced by p . aeruginosa ) and surfactins ( produced by bacillus ssp .). polyethylene pyrolysis product ( pe pyrolysis wax ) was supplied by cynar plc ltd , portlaoise , ireland and was generated from waste agricultural pe films . the strains employed were obtained from the collection of institut pasteur ( cip ), german collection of microorganisms and cell cultures ( dsmz ), agricultural research service culture collection ( nrrl ), american type culture collection ( atcc ), national institute of technology and evaluation , japan ( nbrc ) and private culture collection of f . rojo . the minimal salt medium ( msm ) was prepared as previously described ( schlegel , et al ., 1961 ) and used as the growth medium for all of the culture techniques discussed here . strains were maintained on msm solidified with 15 g l − 1 and supplemented with 20 mm sodium gluconate as a carbon source . pyrolysed polyethylene wax ( pe pyrolysis wax ) was used as a carbon source in all in this specification , the term “ polyhydroxyalkanoate ” experiments . cultures were supplemented with 0 . 05 % w / v rhamnolipids produced by pseudomonas aeruginosa gl - 1 strain as described by arino , et al . ( 1998 ). briefly , p . aeruginosa gl - 1 was grown in a synthetic medium supplemented with 30 g l − 1 of glycerol for 5 days . supernatant was clarified by centrifugation , deproteinised at 100 ° c . for 10 minutes and acidified to ph 2 with 6m hcl . this was followed by an overnight extraction of rhamnolipids with ethyl acetate . organic solvent was evaporated under reduced pressure in order to obtain a honey - like rhamnolipid mixture . composition of rhamnolipids produced was determined accordingly to arino , et al . ( 1998 ) by means of tlc and gc - ms methods . this crude extract was supplemented to the bacterial cultures at a concentration of 500 mg l − 1 ( 0 . 05 % w / v ). for screening purposes , all strains were grown in a 250 ml erlenmeyer flask containing 50 ml of nitrogen limited msm medium and various carbon sources at a final concentration of 1 . 95 gram of carbon per litre ( g c l − 1 = 0 . 05 % w / v ). the flasks were incubated at 30 ° c . with shaking at 250 rpm for 48 hours . to screen for organisms capable of pha accumulation the inorganic nitrogen source ammonium chloride ( nh 4 cl ) was limited to 0 . 25 g l − 1 ( 65 mg n l − 1 = 4 . 6 mm ). in order to establish effect of carbon concentration on growth and pha accumulation , pe pyrolysis wax concentration was increased from 0 . 05 % to 2 % w / v and incubation time was extended up to 6 days . in addition we investigated the effect of two different nitrogen sources ( nh 4 cl and ( nh 4 ) 2 so 4 ) on growth and pha accumulation . the polymer content of lyophilized whole bacterial cells was determined by subjecting cells to acidic methanolysis according to previously described protocols ( brandl , et al ., 1988 , lageveen , et al ., 1988 ). the 3 - hydroxyalkanoic acid methyl esters were assayed by gas chromatography ( gc ) using an agilent 6890n chromatograph equipped with a bp21 capillary column ( 25 m × 0 . 25 mm × 0 . 32 μm ; sge analytical sciences ) and a flame ionization detector ( fid ) with a temperature program of 120 ° c . for 5 min ; followed by ramp of 3 ° c . min − 1 until 180 ° c . held for 10 min . for the peak identification , commercially available 3 - hydroxyalkanoic acids were methylated as described above for pha samples . pha monomer determination was confirmed using an agilent 6890n gc fitted with a 5973 series inert mass spectrophotometer ( ms ), a hp - 5 column ( 12 m × 0 . 2 mm × 0 . 33 μm ; hewlett - packard ) was used with an oven method of 50 ° c . for 3 min , increasing by 10 ° c . min − 1 to 250 ° c . and holding for 1 min . the concentration of nitrogen in the growth media was monitored over time using the previously described indophenol method ( scheiner , 1976 ). bacterial cultures grown in liquid media on pe pyrolysis wax were extracted at various time points with chloroform in order to track the substrate utilisation . the organic layer was analysed on an agilent 6890n series gc fitted with a 5 m × 0 . 53 mm × 0 . 15 μm db - ht sim dis column ( agilent ) using a split mode ( split ratio 2 : 1 ). the oven method employed was 30 ° c . for 1 min , ramping at 7 . 5 ° c . min − 1 to 100 ° c ., followed by a ramp of 10 ° c . min − 1 to 300 ° c . and held at this temperature for 2 min . for peak identification , single hydrocarbons and two alkane standard solutions the 1 st with c8 to c20 and the 2 nd with c21 to c40 ( sigma ) were used . hydrocarbon determination was confirmed using an agilent 6890n gc fitted with a 5973 series inert mass spectrophotometer , a hp - 5 column ( 12 m × 0 . 2 mm × 0 . 33 μm ) ( hewlett - packard ) was used with an oven method of 60 ° c . for 6 min , increasing by 10 ° c . min − 1 to 300 ° c . and holding for 10 min . all nine strains were capable of growing on pe pyrolysis wax and under the screening conditions ( 0 . 05 % pe pyrolysis wax and 0 . 25 g l − 1 ammonium chloride ) two strains accumulated pha ( table 1 ). p . aeruginosa gl - 1 accumulated 3 times more pha than p . aeruginosa pao - 1 ( table 1 ). both strains accumulated mcl - pha i . e . monomers with a carbon chain length ≧ 6 carbons . ( r )- 3 - hydroxydecanoic acid was the predominant monomer accumulated by p . aeruginosa gl - 1 , with ( r )- 3 - hydroxynonanoic acid as the second most prevalent monomer ( fig1 ). pha accumulated by strain pao - 1 contained monomers ranging from ( r )- 3 - hydroxyheptanoic acid to ( r )- 3 - hydroxydodecanoic acid , with ( r )- 3 - hydroxydecanoic acid as the predominant monomer ( fig2 ). p . aeruginosa gl - 1 , which accumulated the most pha from pe pyrolysis wax , was submitted for detailed pe pyrolysis wax hydrocarbon utilisation studies in shake flasks . growth on pe pyrolysis wax was characterised by a lag period of 21 hours . however 58 . 2 % of the pe pyrolysis wax substrate was removed from the growth media by that time . during a 48 h experiment this strain utilised 84 . 4 % of the hydrocarbons supplied ( fig2 ), producing 0 . 23 g l − 1 of cdw of which 9 . 8 % was mcl - pha when grown in msm medium with limited nitrogen to promote pha accumulation . this corresponds to a yield of biomass to substrate ( y x / s ) of 0 . 14 g g c − 1 and yield of pha ( y pha / s ) of 0 . 013 g g c − 1 . increasing the pe pyrolysis wax concentration to 2 % ( w / v ) did not result in better growth of p . aeruginosa gl - 1 or pao1 and no pha accumulation was observed for either strain after 48 hours of growth ( fig3 a and 3b ). p . oleovorans failed to grow with 2 % ( w / v ) pe wax after 48 hours of incubation ( data not shown ). an extended incubation of strain gl - 1 with 2 % ( w / v ) pe wax resulted in similar growth but 1 . 6 fold more pha accumulation ( 4 days ) compared to growth with 0 . 05 % wax ( 2 days ) ( fig3 a ). a dramatic improvement in pha accumulation was observed for strain pao1 with almost 25 % of the cell dry weight as pha ( 5 days ) ( fig3 b ). p . oleovorans failed to grow with 2 % w / v pe pyrolysis wax even after 6 days of incubation ( data not shown ). to further improve the pe wax to pha process , the nitrogen source from ammonium chloride ( nh 4 cl ) to ammonium nitrite ( nh 4 no 2 ) as the latter is known to increase surfactant ( rhamnolipid ) production in p . aeruginosa gl - 1 , which could aid growth on long chain hydrocarbons ( arino , et al ., 1998 ). for both strains , this inorganic nitrogen source enhanced the growth and triggered pha accumulation one day earlier for strain gl - 1 and 2 days earlier for pao1 with 2 % ( w / v ) pe wax . however the level of pha accumulated (% cdw ) did not increase compared to experiments with nh 4 cl . finally , exogenous rhamnolipids were added to the liquid media in order to further enhance growth and pha accumulation . the addition of 0 . 05 % rhamnolipids to the culture media resulted in maximum biomass and pha accumulation in strain pao1 after 2 days of incubation ( fig3 c ) which was an improvement , compared to cells grown in the absence of rhamnolipid where no pha accumulation occurred after 2 days of growth ( fig3 b and 3c ). monomer composition of pha was not affected by the change of nitrogen source or the presence of rhamnolipids ( data not shown ). all microorganisms which were able to grow on pe pyrolysis wax ( 0 . 05 % w / v , nh 4 cl ), but failed to produce pha ( table 2 ), were retested at pe pyrolysis wax concentration of 2 % w / v , ammonium nitrate as the inorganic nitrogen source , and in the presence of 0 . 05 % rhamnolipids four strains showed not only improvement in biomass but were able to accumulate pha ( table 3 ). the two a . calcoaceticus strains , namely bd413 and rr8 , improved biomass levels 1 . 2 and 2 . 7 fold respectively and both accumulated low amounts of pha ( from 2 . 2 to 4 . 1 % cdw , respectively ). p . aeruginosa rr1 improved cdw levels 1 . 5 fold and accumulated 5 . 8 % of the cell dry weight as pha . b . cepacia rr10 achieved 3 . 1 fold higher biomass under the new growth conditions and accumulated 6 . 7 % ( cdw ) pha . none of the strains grew in control flasks where rhamnolipid , but no pe pyrolysis wax , was supplied alone to the growth medium . the composition of pha was very similar to that isolated from strains gl - 1 and pao - 1 with ( r )- 3 - hydroxydecanoic acid as the predominant monomer but both even and uneven carbon chain monomers appearing in the pha ( data not shown ). note - microorganisms were not able to accumulate biomass from rhl as a sole carbon and energy source at this concentration ; 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