JP2008500424A - Method for promoting biodegradation of aliphatic-aromatic copolyesters by enzymatic treatment - Google Patents

Abstract

  Provided is a method for promoting biodegradation of an aliphatic-aromatic copolyester containing more than 60 mol% aromatic acid, based on the total diacid in the copolyester. The method includes contacting the copolyester with at least one hydrolase in an aqueous solution.

Description

  The present invention relates to the field of polymer chemistry. More specifically, the present invention relates to a waste product produced by enzymatic treatment before or after the disposal of an aliphatic-aromatic copolyester having an aromatic acid of more than 60 mol%, based on the total acid content. The present invention relates to a method for promoting biodegradation.

  Synthetic polymers such as polyethylene, polystyrene, and polypropylene have a main chain of only carbon atoms because they must retain their functionality over a wide range of conditions such as temperature and pressure fluctuations, flame exposure, etc. So that it is resistant to chemical and enzymatic degradation. By introducing heteroatoms into the polymer backbone, functional groups such as esters and amines are generated, thereby increasing the hydrolysis of the polyester, i.e., the susceptibility to degradation, resulting in biodegradation of the polyester upon disposal. Ability is improved.

  Polyester polymers are susceptible to hydrolysis by either chemical or enzymatic treatment. Many polyesters, particularly polyesters composed solely of aliphatic monomers, such as polyhydroxybutyrate and poly (ε-caprolactone) are considered biodegradable. Muller et al. (2001) J. MoI. Biotechnol. 86: 87-95; Abou-Zeid et al. (2001) J. MoI. Biotechnol. 86: 113-126. However, aliphatic polyesters lack the desired material properties, particularly durability, for many applications because of their low melting temperatures and high ease of degradation. On the other hand, aromatic polyesters exemplified by polyethylene terephthalate (PET) have desirable durability for use in a wide range of applications, but are generally considered non-biodegradable. Kint, D.C. and Munoz-Guerra, S. et al. (1999) Polym Int 48: 346-352. The tension between durability and biodegradability is always a problem with achieving cost-effective waste disposal. On the other hand, disposable products made with aliphatic polyesters are environmentally attractive but still lack acceptable durability. On the other hand, products made with aromatic polyesters have a favorable robustness, but their disposal is even more ecologically burdensome.

  A partial solution to this dilemma is the use of aliphatic-aromatic copolyesters that produce durable and biodegradable products. However, the use of high aromatic content copolyesters in disposable products is still not completely satisfactory since the biodegradation rate is proportional to the content of aromatic acids in the copolyester. In essence, the tension between durability and biodegradability still remains: the higher the aliphatic content, the higher the biodegradability, but the lower the durability of the product. Furthermore, the higher the aromatic content, the higher the industrial utility, but the higher the possibility of environmental harm.

  Some background in current waste treatment practices is needed as to whether the use of untreated aliphatic-aromatic copolyesters remains a poor solution. The two main waste treatment practices include landfill use and composting. The simplest landfill dumps non-biodegradable or relatively difficult to disassemble materials into specially structured holes that are lined with plastic and / or clay and covered with mud when full Including that. Landfilling is also known as solid waste treatment. In landfills, solid waste is separated from groundwater and air; waste remains dry and generally not subject to microbial action and therefore decomposes very slowly. Ultimately, the use of landfills, where economic and environmental losses continue to increase, is not a sustainable, long-term solution. For example, even if a non-biodegradable aromatic polyester such as PET could be chemically decomposed at the landfill, such decomposition would involve significant energy input from the point of high temperature and break the polymer backbone. Because strong acids are required, the surrounding soil and groundwater need to be potentially purified.

In particular, composting of domestic waste is an ecologically attractive alternative to landfill, but generally requires high costs because it requires the sorting and / or separation of waste first. DeWilde, B., describes a realization effort in Germany that requires the first resource-separated waste to be collected. and Boelens (1998) J. et al. , Polymer Degradation and Stability, 59: 7-12. Composting involves biodegradation of materials by microbial action. It is a two-stage enzyme that occurs mainly during weeks to months of exposure under mild anaerobic conditions, typically below 70 ° C., average temperatures around 55-60 ° C. and 100% relative humidity Is a process. The first stage of polymer biodegradation in compost occurs by hydrolysis of the polyester backbone achieved by extracellular hydrolase. These are usually secreted by a mixture of microflora and cleave the polyester backbone into small polymer fragments and / or respective monomers. Ultimately, cleavage results in fragments and / or monomers that can be absorbed by one or more species of microbiota that are the same or not the same as the microflora that secreted the enzyme. The second stage is the biological stage; microorganisms absorb the fragments and / or monomers and metabolize them into biomass, biogas containing CO ₂ and leachate.

  In addition to serving as an alternative to landfill use, composting can also be used as a landfill aid to reduce the amount of landfill solid waste that cannot be recycled and to produce inexpensive, useful fertilizers. Is done. However, one limitation with respect to compost sales is the visible contamination of undegraded biodegradable polymers, such as plastic films and fiber fragments. Since composting is an environmentally attractive waste process that can produce commercially valuable products, degradation of biodegradable polymers, especially aliphatic-aromatic copolyesters, during composting There is a need for a way to improve. Such a method would increase the environmental and commercial benefits of composting by reducing the need and cost of first separating the waste.

  Other waste treatment implementations include wastewater treatment systems, septic systems and waste treatment systems. Similar to landfill and composting, wastewater treatment is performed at the community level and includes sewage treatment and wastewater reclamation before water is reused or before water enters the body of water. In sewage pre-treatment, diapers and other copolyester products, sand, gravel, large meals from garbage disposal, etc. are screened from untreated waste and miscellaneous wastewater. Thereafter, the collected garbage is processed in a landfill and / or composting system. Other types of settled organic waste recovered from wastewater systems include sludge and scum. These contain copolyesters that are biodegradable. As it settles from the wastewater, sludge and scum are injected into the digestion tank where microorganisms decompose the waste for about a month, which is then sent to the landfill. Septic systems and waste disposal systems are implemented at individual residence levels and are generally not related to the treatment of goods made of highly aromatic copolyesters, such as carbonated beverage containers or diapers. The processing of such articles in these systems generally results in significant damage.

  Certain aliphatic-aromatic copolyesters are biodegradable under mild anaerobic composting conditions, and also have some desirable material properties such as gas barrier permeability, strength, chemical resistance, and sterility. Hold (Muller et al., Supra). Specifically, aliphatic-aromatic copolyesters having up to 60 mol% aromatic acid based on the total acid content are reported to be biodegradable. Goda et al. (2002) Biotechnol Prog 18: 927-934; Witt et al. (1999) Angew Chem Int Ed 38 (10): 1438-1442; Kleeberg et al. (1998) Appl. Env. Microbiol. 64 (5): 1731-1735; Muller et al. (Supra); Koch et al., US Pat. No. 6,255,451. These copolyesters are therefore excellently compostable. On the other hand, high aromatic content copolyesters, that is, copolyesters having an aromatic acid greater than 60 mole percent, based on the total content of acids, are generally “very much such materials would not be suitable for degradation in the composting process It is believed to have a "low" (Muller et al., Supra) biodegradation rate.

  Various methods have been reported to increase the rate of degradation of aliphatic-aromatic copolyesters using hydrolases and / or various microbial aggregates to mineralize polymers into carbon dioxide, biomass, and leachate. Has been. These reports are: Treatment of food waste using hydrolases such as lipases, esterases, proteases, amylases, cutinases, etc. (US Pat. No. 6,350,607 by Cooney, Jr.); The use of enzyme-catalyzed and / or mixed cultures of microorganisms (US Pat. No. 5,990,266 and US Pat. No. 6,191,176 by Tadros et al.); Abou-Zeid et al., Witt et al., Supra; Muller et al., Supra; Kint and Munoz-Guerra, supra; Kleeberg et al., Supra; US Pat. No. 6,255,451 by Koch et al .; Goda et al., Supra. However, these reports use a copolymer having an aromatic acid content of 60 mol% or less, based on the total acid content. None of these reports describe a method for effectively degrading high aromatic content copolyesters.

  The technical problem of composting aliphatic-aromatic copolyesters with aromatic acids above 60 mol%, based on the total acid content, is exemplified by obstacles in the biodegradation of polyethylene terephthalate (PET). In theory, the second stage of enzymatic action in composting can be subjected to PET and thus biodegradable. That is, the microflora can catabolize monomers containing PET aromatic polyester. Actually, microorganisms that convert terephthalic acid into minerals have been isolated from various environments. Bramucci et al. (2002) Appl Microbiol Biotechnol, 58: 255-259, and Junker, F. et al. and Cook, A.A. (1997) Appl Environ Microbiol 63: 2403-10. However, the difficulty in composting PET is in the first stage, not the second stage of enzymatic action; that is, microbially produced hydrolases that result in cleaved fragments and monomers that the microorganism can mineralize In the degradation of the polymer main chain. Since PET inherently contains 100% aromatic diacids (terephthalic acid and its esters) based on the total acid content, the PET polymer backbone is resistant to hydrolysis.

  Modification of the polymer backbone to increase its ease of hydrolysis enhances degradation of aliphatic-aromatic copolyesters containing greater than 60 mole percent terephthalic acid or other aromatic acids, based on the total acid. It is an important key. Modification can be done in several ways. One method is to incorporate molecules into the polyester backbone that can affect biodegradability, such as 5-sulfoisophthalate. See US Pat. No. 6,368,710 (Hayes). This modification makes the polymer less resistant to hydrolysis due to the activating action exerted by the strong electron withdrawing substituents. Kint, D.C. , And Munoz-Guerra, S. See above. Even though it has been shown that 5-sulfoisophthalate-containing aliphatic-aromatic copolyesters can be composted, sulfonated aliphatic-aromatics having more than 60 mol% aromatic acid based on total acid content There remains a need for methods to increase the degradation rate of group copolyesters.

  A second method for increasing the ease of hydrolysis is to treat the highly aromatic copolyester with a hydrolase before or after the copolyester enters the waste cycle. Many enzymes known in the art can degrade polymers containing hydrolyzable groups such as esters, amides and the like. US Pat. No. 6,255,451 (Koch et al.) Includes a cutinase from Humicola insolens and an Aspergillus niger, Mucor Miehei (Lipozyme 20,000 L), and a lipase from Candida antartica (lipase component B). A method for degrading a substrate polymer which is an aliphatic polyester, an aromatic polyester amide or a partially aromatic polyester urethane is described. US Pat. No. 6,066,494 (Hsich) and US Pat. No. 6,254,645 (Kellis et al.) Use lipase or polyesterase to modify polyester fibers to produce A method for increasing the wettability and absorbency of the is described. US Pat. No. 6,350,607 (Cooney, Jr.) describes the use of an enzyme for treating macerated food waste in combination with a waste disposal device; US Pat. No. 5,464,766 (Bruno) reports a waste treatment composition containing bacteria and enzymes for use in domestic waste and garden waste.

  However, none of these reports is related to the degradation of aliphatic-aromatic polymers having an aromatic acid content of more than 60 mol%, based on the total acid content. In order to find a biodegradation solution in the current waste context, especially in compost, despite the inability to biodegrade highly aromatic copolyesters, these are just It is necessary to make the composting of local governments a feasible waste process. Such a solution eliminates the need for waste separation of resources and provides commercially valuable fertilizer quality compost. Furthermore, such a solution will also accelerate the degradation rate of highly aromatics discarded in landfills.

  Thus, the problem to be solved is an aliphatic-aromatic copolyester having an aromatic acid content of greater than 60 mole percent based on the total acid content in general composting conditions and other waste contexts. Is the development of a method to increase the biodegradation rate. The solution provided is an effective enzymatic treatment of the disposable copolyester that initiates and promotes biodegradation either before or after the copolyester is discarded. In contrast, the solution is an effective enzymatic treatment of disposable copolyesters that has long been regarded as biodegrading very slowly, which results in an initial stage of biodegradation, namely hydrolysis of the polymer backbone. Is induced and promoted. The solution of the present invention is particularly useful for promoting the degradation of sulfonated copolyesters having aromatic acid content greater than 60 mol%.

The present invention provides a method for enhancing hydrolysis of an aliphatic-aromatic copolyester by microorganisms, the copolyester comprising:
At least one aromatic dicarboxylic acid or ester thereof;
At least one aliphatic dicarboxylic acid or ester thereof;
And the aromatic acid comprises more than 60 mol% and up to about 99 mol% of the total dicarboxylic acid in the copolyester,
The method includes contacting the copolyester with at least one hydrolase in aqueous solution. Such copolyesters are also identified as highly aromatic copolyesters or subject copolyesters. More specifically, the subject copolyesters contain at least about 61 mol% to about 90 mol%, or at least about 70 mol% of at least one aromatic dicarboxylic acid or ester, based on the total dicarboxylic acid in the copolyester. Or 75 mol%, or 80 mol%, up to about 90 mol%.

  In one embodiment, the at least one aromatic dicarboxylic acid or ester is terephthalic acid, dimethyl terephthalate, isophthalic acid, dimethyl isophthalate, 2,6-naphthalenedicarboxylic acid, dimethyl-2,6-dicarboxylic acid, dimethyl-2. , 6-naphthalene and mixtures thereof, the at least one aliphatic dicarboxylic acid or ester is succinic acid, dimethyl succinate, glutaric acid, dimethyl glutarate, 2-methylglutaric acid, 3-methyl It is selected from the group consisting of glutaric acid, adipic acid, dimethyl adipate, oxalic acid, dimethyl oxalate, malonic acid, dimethyl malonate, and mixtures thereof. In this embodiment, the copolyester may comprise from about 1 mol% to less than about 40 mol% of at least one aliphatic acid or ester, based on the total dicarboxylic acid in the copolyester. In other embodiments, the copolyester may comprise from about 10 mol% to less than 40 mol% of at least one aliphatic acid or ester. In further embodiments, the copolyester may comprise about 20 mol% to less than 40 mol% of at least one aliphatic acid or ester. In these embodiments, the copolyester further comprises 1,2-ethanediol, 1,3-propanediol, 1,4-butanediol, 1,6-hexanediol, ethylene glycol, di (ethylene glycol), tri ( Ethylene glycol), poly (ethylene glycol), poly (alkylene ether) glycol, poly (propylene ether) glycol, and mixtures thereof may include at least one diol. In other embodiments, the copolyester is combined with starch, protein, cellulose, wax, filler, silicate filler, polylactic acid, polyhydroxyamide, or mixtures thereof.

  The copolyester may also include a sulfonated compound comprising from about 0.1 to about 10.0 mole percent of the copolyester, based on the total diol or total dicarboxylic acid in the copolyester. In other embodiments, the copolyester may comprise about 0.1 to 5.0 mole percent of the sulfonated compound. The sulfonated compound is selected from the group consisting of sulfonated mono- or dicarboxylic acids, or esters or metal salts thereof. The sulfonated compound is further selected from the group consisting of sulfosuccinic acid, 3-sulfobenzoic acid, 4-sulfobenzoic acid, 5-sulfosalicylic acid, sulfophthalic acid, sulfoterephthalic acid, and 5-sulfoisophthalic acid, or esters or salts thereof. Is done. In this embodiment, the sulfonated copolyester comprises from about 1 mole percent to less than about 40 mole percent of at least one aliphatic carboxylic acid or ester, in other embodiments, from about 10 moles, based on the total dicarboxylic acid content. % To less than about 40 mol%; it may further comprise at least one diol. In other embodiments, the sulfonated copolyester can comprise from about 20 mole percent to less than about 40 mole percent of at least one aliphatic carboxylic acid or ester. In another embodiment, the copolyester comprises about 17.5 mol% dimethyl glutarate, about 2 mol% dimethyl 5-sulfoisophthalate, sodium salt based on the total dicarboxylic acid in the copolyester, and further comprises poly (ethylene Glycol) may comprise about 8% by weight.

  In other embodiments, the aqueous solution comprises about 0.1 to about 10% by weight of at least one hydrolase selected from the group consisting of proteases, lipases, cutinases, esterases, and combinations thereof. In any of the above embodiments, the at least one hydrolase comprises a lipase.

  In one embodiment, at least one hydrolase is contacted with the copolyester prior to degradation of the copolyester by the microorganism. In other embodiments, contact is made when the copolyester is in a waste context that is a solid waste or compost or wastewater treatment system. Contact of the subject copolyester with at least one hydrolase is by spraying, painting, coating, applying, or mixing.

  The present invention can be more fully understood from the drawings and detailed description together with which this application forms.

  In a broad sense, the present invention provides a method for promoting biodegradation of aliphatic-aromatic copolyester waste. Specifically, the present invention provides a method for increasing the biodegradation rate of a highly aromatic copolyester by treatment with a hydrolase. Such treatment promotes the overall biodegradation of the highly aromatic copolyester by increasing the degradation of the polymer backbone. In a particularly useful embodiment, the high aliphatic-aromatic copolyester includes a sulfonated component.

In this disclosure, a number of terms and abbreviations are used according to the following definitions:
By “high aromatic copolyester” is meant an aliphatic-aromatic polyester that contains more than 60 mol% aromatic diacid, such as terephthalic acid, based on the total acid content of the polyester. These are also referred to as “copolyesters of interest”. Particularly useful highly aromatic copolyesters include a sulfonated component.

  The term “aliphatic-aromatic copolyester” means a polyester in which a part of the aliphatic diacid structural unit is substituted with an aromatic diacid.

  “Diacid content 60 mol%” means “dicarboxylic acid content 60 mol%” and is considered equal. Also, “acid content 60 mol%” means “diacid content 60 mol%” as in this description, and the aliphatic-aromatic polyester contains an aliphatic dicarboxylic acid and an aromatic dicarboxylic acid. .

  “Total acid content” and “total dicarboxylic acid” equally mean the total content of aromatic and aliphatic dicarboxylic acids in the aliphatic-aromatic copolyester.

  “Sulfonated copolyester” means a polyester containing a 5-sulfoisophthalic acid derivative as one of the constituent monomers.

  “Sulfonated aliphatic-aromatic copolyesters” contain more than 60 mol% aromatic dicarboxylic acids, generally terephthalic acid; less than 40 mol% aliphatic dicarboxylic acids such as glutaric acid, adipic acid and succinic acid. Specifically, 0.1 to 10 mol% of a sulfonated component such as 5-sulfoisophthalic acid, which is 0.1 to 3 mol% of dimethyl-5-sulfoisophthalic acid sodium salt, and optionally 0. From 1 mol% to 5.0 mol%; optionally, diols such as ethylene glycol, 1,3-propanediol, and 1,4-butanediol.

  "Hydrolyzing enzyme" means a type of hydrolase such as, but not limited to, protease, lipase, cutinase and esterase. These enzymes have proven useful for a variety of industrial applications, such as enzymatic hydrolysis of polyesters.

  “Hydrolysis” means the main mechanism of action in polymer degradation, and as used herein means hydrolysis mediated by hydrolase. It cleaves the ester and amide bonds from the polymer backbone by the addition of water mediated by hydrolase, resulting in the parent carboxylic acid group and the respective functional group, ie the hydroxyl function for the ester. (Ie alcohols such as methanol) and the generation of suitable amines for amides. This is also known as hydrolysis of the polymer backbone.

  “Lipase” means an enzyme that catalyzes the hydrolysis of fatty acids to glycerol and fatty acids by hydrolyzing ester bonds.

“Cutinase” means a hydrolase that degrades cuticle, a cuticle polymer derived from higher plants, which is a polyester composed of hydroxy and epoxy fatty acids. Kuching fatty acids are usually n-C ₁₆ and n-C ₁₈ and contain 1 to 3 hydroxyl groups. Although ester linkages predominate in cutin, peroxide bridges and ether linkages can also be present.

  “Esterase” and “polyesterase” are used interchangeably to refer to an enzyme that catalyzes the hydrolysis of an ester.

  “Protease” means an enzyme that catalyzes the hydrolytic breakdown of proteins by hydrolysis of peptide bonds.

  "Biodegradable" or "degradable" describes a polymer that can be chemically degraded by natural biological processes, such as being digested by bacteria or fungi into small components that are not harmful to the environment. To do.

  By “biodegradation by microbial action” or “biodegradation” is meant what is considered to be a two-step process of degrading copolyesters that are discarded as waste. In the first stage, a hydrolase secreted by soil microorganisms degrades the polymer backbone of the copolyester into monomers and / or small fragments; in the second stage, the same or different microorganisms as in the first stage To break down the fragments and catabolize them into biomass, biogas and leachate.

  “Biodegradation rate / biodegradability” means the rate, rate or rate at which biodegradation occurs. This depends on conditions such as temperature, humidity, exposure to air, etc. in the waste context in which the target copolyester is discarded. Comparison of biodegradation rates among various waste contexts is generally sequential and relative. Because biodegradation is a specific indicator of specific waste conditions and specific waste quantities, the absolute rate of biodegradation for a specific waste context is difficult to calculate accurately.

  “Waste context” means and encompasses composting, landfills, solid waste, wastewater treatment systems, septic tank systems, and refuse treatment systems.

  “Composting” means decomposing waste at a relative humidity of nd00% or near 70 ° C. or less and an average of 55 to 60 ° C. for a period ranging from 2 weeks to several months or more. Means process. The waste continues to be broken down into low molecular weight fragments and / or monomers and eventually biodegraded, that is, completely metabolized by the microorganisms into biomass, biogas, and leachate.

  By “compost tea” is meant a liquid (aqueous) extract from compost containing soluble nutrients and a diverse mixture of microorganisms that can grow when the compost material is spoiled.

  A “wastewater treatment system”, also known as sewage treatment, includes a multi-step process that regenerates wastewater before it enters the body of water again, or before it is applied to land or reused. In general, the first stage involves screening solid waste that may contain the target copolyester and generally accumulates in the landfill. At a later stage, solid organic waste containing accumulated sludge and scum, which may also contain the copolyester of interest, is digested in a digestion tank for about a month. It is contemplated to contact the hydrolytic enzyme with both the sorted solid waste and the material in the digestion tank.

  “Landfill” means a solid waste that is isolated from groundwater and air and kept dry, thereby preventing the decomposition of waste by microbial action, resulting in slow decomposition of waste It means a specially structured solid waste hole lined with plastic and / or clay into which the object is dumped. A landfill is a solid waste context at ambient air or relatively adjacent soil temperature at a relatively low humidity, typically in an anaerobic environment.

  “Contacting” is equivalent to “treating” and means placing the hydrolase in contact with the copolyester so that the hydrolase can initiate hydrolysis of the polymer backbone. To do. “Treatment” means contacting the hydrolase with the copolyester at any point during the copolyester use and disposal cycle, and “pretreatment” of the copolyester before disposal and after disposal. Includes the concept of “post-processing”. “Contacting” includes spraying, treating, pouring in or on, mixing, combining, painting, coating, applying, attaching, and otherwise transmitting hydrolase with the copolyester.

  By “other biodegradable compounds” is meant, in addition to aliphatic-aromatic copolyesters, substances that can decay naturally, harmlessly, and relatively quickly than high aromatic copolyesters. These materials include starch, protein, cellulose, beeswax, montanic ester wax, leather, paper, textiles, fillers such as calcium carbonate, calcium phosphate, and titanium dioxide, cellulosic fillers, silicon dioxide, etc. Silicate fillers, biodegradable adhesives, polylactic acid, polyhydroxyamides and mixtures thereof.

All other terms can be found in the following dictionaries: WEBSTER'S THIRD NEW INTERNATIONAL DICTIONARY, UNABRIDGED (1993), Merriam-Webster, Springfield, MA, and LEWIDENY RID (2001) , John Wiley & Sons, New York, NY.

Abbreviation

Structure of Aliphatic-Aromatic Copolymers Biodegraded by the Method of the Invention The method of the invention increases the biodegradation rate of disposable polymers by treating the polymer with a hydrolase catalyst. Polymers are used to produce a wide variety of industrial products, including doughs, containers and packaging, because they can be formed into products at high temperatures and exhibit gas barrier and biodegradable properties. .

A meaningful polymer for this process is a copolyester containing ester groups in the chain derived from the condensation of a diacid and a diol or from the polymerization of a hydroxy acid. Copolyester is a biodegradable polymer because it retains functional groups such as esters, amides, and urethanes that are easily hydrolyzed, that is, easily hydrolytically cleaved. Monomers that can cleave the polymer backbone by hydrolysis of these functional groups mediated by hydrolases, and can be absorbed by microorganisms and then metabolized into biomass, biogas (ie, CO ₂ ), and leachate Small polymer fragments are produced.

  In aliphatic polyesters, the diacid component is primarily an aliphatic acid; these polymers are exemplified by polyhydroxyalkanoates produced by microorganisms, or synthetic polymers such as polycaprolactone or polylactide. Aromatic polyesters are polymers such as polyethylene terephthalate (PET) or polybutylene terephthalate (PBT) in which the diacid component is an aromatic acid such as terephthalic acid or an ester thereof. Many of the preferred material properties of polyesters, such as thermal stability, gas and liquid permeability, are related to the relative amount of aromatic monomers in the polyester. The aromatic polyester constitutes a disposable plastic beverage bottle, for example. Since aromatic acids used, such as terephthalic acid, make the polymer backbone resistant to hydrolysis, they are generally considered difficult or impossible to biodegrade.

  Hydrolysis is a process in which molecules are broken into two by adding water molecules. This process can be performed both chemically and enzymatically. In a hydrolysis reaction involving cleavage of an ester bond, one hydrolysis product contains a hydroxyl functional group and the other hydrolysis product contains a carboxylic acid functional group. Amides hydrolyze to the parent carboxylic acid and the corresponding amine.

Aliphatic - aromatic copolyesters, aromatic diacids derived, for example, terephthalic acid or an ester derivative thereof, and generally linear, of a mixture of aliphatic diacids are polyalkylene group length C ₂ -C ₁₀ Is done. Muller et al. (Supra) are essentially non-biodegradable (or at least degrade) aliphatic-aromatic copolyesters containing more than 60 mole% terephthalic acid as diacid, based on the total acid content. Are very slow) and are therefore considered noncompostable.

  Aliphatic-aromatic copolyesters containing more than 60 mol% terephthalic acid, based on the total acid content, and aliphatic-aromatic copolyesters also having sulfonate radicals of 0.1-10 mol% (hereby incorporated by reference U.S. Pat. No. 5,097,004 (Galllagher) and U.S. Pat. No. 6,368,710 (Hayes)) are designed to address that. Copolyester. Even though these copolyesters are reported to be biodegradable, it is generally necessary to increase their biodegradability. The technical solution of the method of the present invention is to contact the sulfonated copolyester with a hydrolase either before or after disposal to increase the hydrolysis rate of the polyester backbone. In this way, the biodegradability of these copolyesters is enhanced in various waste contexts such as composting facilities, solid waste facilities, and wastewater treatment systems.

  Aliphatic dicarboxylic acid components include unsubstituted, substituted, linear and branched, aliphatic dicarboxylic acids, and lower alkyl esters of aliphatic dicarboxylic acids having 2 to 36 carbon atoms. Specific examples of the aliphatic dicarboxylic acid component include oxalic acid, dimethyl oxalate, malonic acid, dimethyl malonate, succinic acid, dimethyl succinate, methyl succinic acid, glutaric acid, dimethyl glutarate, 2-methylglutaric acid, 3-methylglutaric acid, adipic acid, dimethyl adipate, 3-methyladipic acid, 2,2,5,5-tetramethylhexanedioic acid, pimelic acid, suberic acid, azelaic acid, dimethyl azelate, sebacic acid, 1 , 11-undecanedicarboxylic acid, 1,10-decanedicarboxylic acid, undecanedioic acid, 1,12-dodecanedicarboxylic acid, hexadecanedioic acid, docosanedioic acid, tetracosanedioic acid, dimer acid, and mixtures thereof It is done. The following succinic acid, dimethyl succinate, glutaric acid, dimethyl glutarate, adipic acid, dimethyl adipate and mixtures thereof are particularly useful. However, these lists are not exhaustive and aliphatic dicarboxylic acid components known in the art may also be useful in the methods of the present invention.

  Aromatic dicarboxylic acid components include unsubstituted and substituted aromatic dicarboxylic acids and lower alkyl esters of aromatic dicarboxylic acids having 8 to 20 carbon atoms. Examples of desirable diacid components include those derived from terephthalate, isophthalate, naphthalene and dibenzoate. Specific examples of the aromatic dicarboxylic acid component include terephthalic acid, dimethyl terephthalate, isophthalic acid, dimethyl isophthalate, 2,6-naphthalenedicarboxylic acid, dimethyl-2,6-naphthalene, 2,7-naphthalenedicarboxylic acid, Dimethyl-2,7-naphthalene, 3,4'-diphenyl ether dicarboxylic acid, dimethyl-3,4'diphenyl ether dicarboxylate, 4,4'-diphenyl ether dicarboxylic acid, dimethyl-4,4'-diphenyl ether dicarboxylate, 3, , 4'-diphenyl sulfide dicarboxylic acid, dimethyl-3,4'-diphenyl sulfide dicarboxylate, 4,4'-diphenyl sulfide dicarboxylic acid, dimethyl-4,4'-diphenyl sulfide dicarboxylate, 3,4'- Zife Sulfone dicarboxylic acid, dimethyl-3,4'-diphenyl sulfone dicarboxylate, 4,4'-diphenyl sulfone dicarboxylic acid, dimethyl-4,4'-diphenyl sulfone dicarboxylate, 3,4'-benzophenone dicarboxylic acid, Dimethyl-3,4'-benzophenone dicarboxylate, 4,4'-benzophenone dicarboxylic acid, dimethyl-4,4'-benzophenone dicarboxylate, 1,4-naphthalenedicarboxylic acid, dimethyl-1,4-naphthalene, 4 4,4'-methylenebis (benzoic acid), dimethyl-4,4'-methylenebis (benzoate), and mixtures thereof. The following aromatic dicarboxylic acid components are particularly useful: terephthalic acid, dimethyl terephthalate, isophthalic acid, dimethyl isophthalate, 2,6-naphthalenedicarboxylic acid, dimethyl-2,6-naphthalene, and mixtures thereof. However, these lists are not exhaustive and aromatic dicarboxylic acid components known in the art may also be useful in the methods of the present invention.

  Sulfonated aliphatic-aromatic copolyesters have been reported in the art. A compostable aliphatic-aromatic copolyester consisting of 5 to about 40 mole percent C2-C12 aliphatic diacid, 1 to 30 mole percent di (ethylene glycol) and tri (ethylene glycol), the remainder At least 85 mol% of the acid component is terephthalic acid, and the remainder of the glycol component is selected from the group consisting of ethylene glycol, 1,3-propanediol, and 1,4-butanediol; US Pat. No. 5,171,308 (Gallerger et al.) Describing copolyesters, wherein 2.5 mol% is composed of sites containing alkaline or alkali metal sulfo groups; Sulfo containing 40 mol%, 0.1 to 2.5 mol% of the component having an alkali / alkali metal sulfo group U.S. Pat. No. 5,171,309 (Galllagher et al.) Describing activated aliphatic-aromatic copolyesters; starch and up to 20 mol% di (ethylene glycol), alkali metal or alkaline earth metal An aliphatic-aromatic copolyester having 0.1 to 15 mol% sulfo groups, 10 to 40 mol% aliphatic diacids such as adipic acid or glutaric acid, ethylene glycol, and 45 to 89.9 mol% terephthalic acid; US Pat. No. 5,219,646 (Galllagher et al.) Describing a compostable product comprising a blend of: a US further having a 0-0.4 mole fraction of polyester derived from a hydroxy acid U.S. Pat. No. 5,295,985 describing a copolyester having the composition of US Pat. No. 5,171,309 (Romesser et al.); US Pat. No. 6,018,004 and US Pat. No. 6,297 which describe biodegradable aliphatic-aromatic copolyesters which may contain 0-5 mol% of sulfonate compounds. 347 (Warzelhan et al.); US Pat. No. 6,368,710 describing sulfonated copolyesters having about 0.1 to about 10 mole percent sulfonated compounds and isosorbide based on the total dicarboxylic acid. See the specification (Hayes).

  The sulfonated composition of the present invention should contain less than 1 to 40 mol% of the aliphatic dicarboxylic acid component based on the total diacid content. In other embodiments, the sulfonated copolyester aliphatic dicarboxylic acid component is less than about 10-40 mol% based on the total diacid content. In other embodiments, the sulfonated copolyester aliphatic acid component is less than about 20-40 mol%. An example of an aliphatic sulfonate component includes a metal salt of sulfosuccinic acid. The sulfonated composition of the present invention should contain more than 60 mol% and up to 99 mol% aromatic dicarboxylic acid component, based on the total diacid content. In other embodiments, the aromatic acid component is greater than 60 mole percent and up to about 90 mole percent based on the total diacid content. In other embodiments, the aromatic acid component is 70-90 mol%, optionally 80-90 mol%, based on the total diacid content. Examples of aromatic sulfonate components useful as end groups include metal salts of 3-sulfobenzoic acid, 4-sulfobenzoic acid, and 5-sulfosalicylic acid.

  Useful sulfonate components are sulfonate components in which the sulfonate group is bonded to an aromatic dicarboxylic acid and the aromatic nucleus can be benzene, naphthalene, diphenyl, oxydiphenyl, sulfonyldiphenyl, methylenediphenyl, and the like. Useful sulfonate monomers include residues of sulfonate substituted phthalic acid, terephthalic acid, isophthalic acid, 2,6-naphthalenedicarboxylic acid. Particularly useful sulfonate components are metal salts of 5-sulfoisophthalic acid or lower alkyl esters of 5-sulfoisophthalate. The metal salt is a monovalent or polyvalent alkali metal ion, alkaline earth metal ion, or other metal ion. The alkali metal ion is sodium, potassium or lithium, and alkaline earth metals such as magnesium are also useful. Other useful metal ions include transition metal ions such as zinc, cobalt or iron. In one embodiment, the sulfo group-containing component is about 0.1 mol% to about 10 mol% of the sulfonated copolyester composition. The process of the present invention is particularly useful when the sulfo group-containing component is at a level of incorporation of 0.1 to 5.0 mole percent within the sulfonated copolyester of the present invention.

Diol components useful in the practice of the present invention include glycols containing 2 to 12 carbon atoms, glycol ethers containing 4 to 12 carbon atoms, structural formula HO (AO) _n H ( _where A is Polyether glycol having 2 to 6 carbon atoms and n is an integer of 2 to 400). In general, such polyether glycols have a molecular weight of about 400-4000. Glycols usually contain 2 to 8 carbon atoms but generally contain 4 to 8 carbon atoms. Some representative examples of glycols that can be used as the diol component include 1,2-ethanediol, 1,3-propanediol, 1,4-butanediol, 1,6-hexanediol, ethylene glycol , Di (ethylene glycol), tri (ethylene glycol), poly (ethylene) glycol, 1,3-propylene glycol, 1,2-propylene glycol, 2,2-diethyl-1,3-propanediol, 2,2- Dimethyl-1,3-propanediol, 2-ethyl-2-butyl-1,3-propanediol, 2-ethyl 2-isobutyl-1,3-propanediol, 1,3-butanediol, 1,5-pentane Diol, 2,2,4-trimethyl-1,6-hexanediol, 1,3-cyclohexanemethanol, , 4-cyclohexane methanol, 2,2,4,4, - tetramethyl-1,3-cyclobutanediol, isosorbide, poly (alkylene ether) glycol, poly (propylene ether) glycol, and the like.

  Other biodegradable compounds can be combined with the aliphatic-aromatic copolyester. These compounds include starch, protein, cellulose, beeswax, montanic ester wax, leather, paper, fabric, fillers such as calcium carbonate, calcium phosphate, and titanium dioxide, cellulosic fillers, silicon dioxide, etc. Silicate fillers, biodegradable adhesives, polylactic acid, polyhydroxyamides and mixtures thereof.

  In one embodiment of the method of the present invention, the aromatic acid comprises more than 60 mol% and up to 99 mol% of the total acid content of the copolyester. In other embodiments, the aromatic acid comprises from 61 mole percent to about 90 mole percent of the total acid content of the copolyester. In other embodiments, the aromatic acid comprises at least 70 mole percent to about 90 mole percent of the total acid content of the copolyester. In other embodiments, the aromatic acid comprises at least 80 mole percent to about 90 mole percent of the total acid content of the copolyester. In still other embodiments, the aromatic acid comprises at least 90 mole percent to about 99 mole percent of the total acid content. In other embodiments, the aromatic acid comprises at least 95 mole percent to about 99 mole percent of the total diacid content of the copolyester. In a further embodiment, the aromatic acid comprises at least 97 mole percent to about 99 mole percent of the total diacid content of the copolyester.

  Of particular importance to the process of the present invention are two types of biodegradable copolyesters, “Polymer A” and “Polymer B”. Polymer A consists of 17.5 mol% DBE-5 (dimethyl glutarate, CAS # 1119-40-0, based on 100 mol% total acid), 2 mol% DRL-6 (dimethyl 5-sulfoisophthalate, sodium A sulfonated aliphatic-aromatic polymer comprising poly (ethylene terephthalate) together with a salt, CAS # 3965-55-7). Polymer B consists of 17.5 mol% DBE-5 (dimethyl glutarate, CAS # 1119-40-0, based on 100 mol% total acid), 2 mol% DRL-6 (dimethyl 5-sulfoisophthalate, sodium A sulfonated aliphatic-aromatic polymer comprising poly (ethylene terephthalate) together with 8% by weight of salt, CAS # 3965-55-7) and poly (ethylene glycol) (1000 MW, bop). The polymer is present as particles, films or laminated.

Process of biodegradation The following: terephthalic acid (abbreviated T): ethylene glycol (abbreviated 2G) or other lower alkylene glycol (such as 3G or 4G); polyethylene ether radical (abbreviated DEG or TEG); indicated molecular weight ( derived from; metal salt of a sulfonated derivative of such and about 0.1 to 5.0 mole% of the amount of 5-sulfoisophthalic acid (abbreviation 5SI); C ₂ -C ₄ polyalkylene ether glycol radicals PAG) Aliphatic-aromatic copolyesters begin to degrade when subjected to high humidity and temperature conditions typically representing compost. Table 1 in Muller et al. (Above) examines the aliphatic component in aromatic copolyesters.

  Biodegradation in composting is considered to be a two-stage process. In the first stage, a hydrolase from microorganisms present in compost, which secretes a hydrolase, is generally applied to solid waste. The secretion rate and overall amount of hydrolase observed under composting conditions is very low for complete biodegradation of aliphatic-aromatic copolyesters. Hydrolysis of the polyester backbone is recognized as the rate-limiting step during composting. Exogenously supplied hydrolase greatly increases the biodegradation rate of aromatic-aliphatic copolyesters. As the amount of enzyme increases, a high concentration of the target copolyester is hydrolyzed to low molecular weight compounds and / or monomers. Microorganisms that can metabolize low molecular weight compounds / monomers then catabolize them into biomass, biogas, and leachate. The microorganism that catabolizes is the same or different from the microorganism that secretes the enzyme. The low molecular weight compound / monomer bulk resulting from degradation of the polymer backbone includes terephthalic acid, aliphatic acids, hydroxy acids, and glycols.

  Various microorganisms are used in the composting process. For example, microorganisms that catabolize terephthalic acid have been reported. Bramucci et al., Appl Microbiol Biotechnol, 58: 255-259 (2002); US Pat. No. 6,187,569 and US Pat. No. 6,461,840 (Bramucci et al.); Junker, F .; and Cook, A.A. , Appl Environ Microbiol, 63: 2403-2410 (1997). The method of the present invention contemplates the use of microorganisms that catabolize low molecular weight compounds resulting from hydrolysis to produce hydrolase and provide complete biodegradation. It is further contemplated to engineer the microorganism to increase hydrolase activity and / or increase the ability to metabolize the resulting low molecular weight compound. Since many of the organisms normally isolated from the composting environment are somewhat thermophilic and efficiently decompose solid waste at temperatures of 55-60 ° C., the process of the present invention is performed at standard composting conditions. In order to optimally grow and decompose solid waste, genetic manipulation of microbial catalysts is also contemplated.

  On the other hand, composting is the accumulation of solid waste in landfills. Traditional landfills, or “dry tomb” landfills, are made in or on soil, where solid waste is isolated from groundwater, air and rain It is a carefully designed structure. Its temperature is that of the surrounding soil or ambient air, the humidity is low, the conditions are generally anaerobic, thereby preventing degradation by microbial action, not months or years in compost, Decomposes relatively slowly over decades and even longer. Unlike that, bioreactor landfills function to convert and decompose solid waste more quickly by adding liquids and air that enhance microbial degradation processes similar to those in composting.

  In drainage, also known as sewage, a treatment system, a common treatment process, involves pumping sewage from a residence to a treatment plant. Wastewater entering the plant is first subjected to sorting of solid waste such as diapers, beverage bottles, films and other copolyester products, sand, gravel, mud, food, etc. and then sent to a landfill. The wastewater is then aerated to remove dissolved gas and the wastewater enters a series of tanks, each of which is divided into two sections. The first section is for aeration; aerating the wastewater with air helps to settle small high-concentration particles that may contain grit, a copolyester material. The grit is pumped out of the tank and transported to the landfill like sorted solid waste. After aeration, water enters the second or settling section where sludge or solid organic waste settles from the wastewater and is subsequently pumped from the tank into the digester. Sludge settles in the sedimentation tank, but light substances called scum and which may contain grease, oil, and copolyester particles float on the wastewater surface. A rake scoops scum from the wastewater surface. Scum is also pumped to the digester with sludge. The digester is a large, heated, closed tank in which sludge and scum are degraded by bacteria for about 18-30 days, generally under anaerobic conditions at a temperature of about 36-40 ° C. The pH in the digestion tank is generally in the range of 6-11. Digested products are dried and generally sent to landfills. The above description is not intended to specify all variations in wastewater treatment systems known to those skilled in the art, but is a broad description.

  Biodegradation by microbial action can occur in most waste contexts; however, the rate at which it occurs depends, inter alia, on the humidity, air content and temperature of the waste context. One skilled in the art can select or design a suitable hydrolase that is optimally used under various waste treatment conditions, i.e., under changing pH, temperature.

Enhanced biodegradation: the method of the present invention The method of the present invention increases the biodegradability of copolyesters that are difficult to biodegrade and is useful in most waste contexts such as composting, landfills and wastewater treatment systems It is. The process of the present invention works by increasing the hydrolysis rate of the polymer backbone of an aliphatic-aromatic copolyester having an aromatic acid of greater than 60 mole percent based on the total acid content. Increased hydrolysis occurs by contacting the copolyester with a hydrolase.

Hydrolytic enzymes in the present invention Hydrolytic enzymes useful in the present invention are known in the art and include in particular esterases, lipases, cutinases, and proteases. (1) Reported in US Pat. No. 5,464,766 (Bruno), which provides a powdered enzyme / bacterial fermentation product to digest and liquefy organic waste streamed into an on-site waste treatment system (2) modification of the polyester surface for stronger adhesion to cationic materials as reported in US Pat. No. 6,254,645 (Kellis et al.). Of the polyesters reported in US Pat. No. 6,255,451 (Koch et al.) And US Pat. No. 6,191,176 (Tadros et al.). With respect to hydrolysis; hydrolytic enzymes are used to modify polyesters.

  Many hydrolases are commercially available. The enzymes used in the examples were commercially available enzymes (Aldrich Chemicals, Milwaukee, WI or Sigma Chemical, St. Louis, MO).

  Instead, hydrolases can be produced biologically. Hydrolytic enzymes have been reported in various organisms such as bacteria, fungi, yeasts, plants and organisms belonging to animals. Examples of suitable species known to produce suitable enzymes include, but are not limited to, Absidia, Aspergillus, Achromobacter, Aureobasidium ( Aureobasidium), Bacillus, Brochotrix, Candida, Chromobacter, Coprinus, Fusarium, Geotricum, Geotricum, Geotricum, Geotricum, Hyphozyma genus, Lactobacillus genus, Penicillium genus, Pseudomonas genus ( Seudomonas), Mucor (Mucor), Rhizomucor genus (Rhizomucor), Rhizopus (Rhizopus), Rhodotorula (Rhodotorula), Sporobolomyces sp., Thermomyces sp., Thiaroporella genus, and Trichoderma (Trichoderma) can be mentioned. Particularly useful are lipases from Candida antarctica (particularly component B), Rhizomucor miehei, Aspergillus niger and Thermomyces lanuginosus.

  Furthermore, the hydrolase can be genetically manipulated. For example, enzyme specificity and catalytic rate can be improved by genetic engineering of the nucleic acid encoding the enzyme using standard recombinant methods in the art. In addition, the gene encoding the desired enzyme can be expressed by standard recombinant techniques in a heterologous host cell for production of the enzyme.

  The hydrolase may be used as a pure enzyme or in the presence of other enzymes and may or may not be immobilized. Furthermore, the enzyme catalyst can take the form of whole cells that express the endogenous catalyst. Alternatively, a mixture of hydrolases can be used to hydrolyze the polyester backbone. Enzymes used in the method of the present invention are typically active at temperatures of 25 ° C. to 70 ° C., more effectively 50 ° C. to 70 ° C., most effectively 55 ° C. to 65 ° C., and are specific to the composting environment. Reflects the conditions. However, those skilled in the art can select hydrolases that function optimally under various waste stream conditions.

  Many enzymes known in the art can degrade polymers containing hydrolyzable groups such as esters, amides and the like. US Pat. No. 6,255,451 (Koch et al.) Describes a cutinase from Humicola insolen for degrading a substrate polymer that is an aliphatic polyester, aromatic polyester amide or partially aromatic polyester urethane. The use of lipases from Aspergillus niger, Mucor Miehei (Lipozyme 20,000 L), and Candida antartica (lipase component B) is taught. In addition, US Pat. No. 6,066,494 (Hsich et al.) And US Pat. No. 6,254,645 (Kellis et al.) Describe polyester fibers to increase fabric wettability and absorbency. The use of lipases or polyesterases for modification is described. The use of enzymes for the treatment of macerated food waste in conjunction with waste treatment equipment is also described in US Pat. No. 6,350,607 (Cooney, Jr.). A waste treatment composition containing bacteria and enzymes for domestic waste and garden waste is reported in US Pat. No. 5,464,766 (Bruno).

Enzymatic treatment: contact of hydrolase with copolyester To increase the biodegradation rate of an aliphatic-aromatic copolyester having an aromatic acid content of more than 60 mol%, based on the total acid content, the present invention This method provides a treatment step in which the copolyester of interest is contacted with one or more hydrolases, either before or after disposal. Specifically, in addition to the biodegradation stage, enzyme treatment: microbial hydrolysis followed by microbial catabolism is outlined above. The enzymatic treatment of the method of the present invention is an additional hydrolysis step for the biodegradation process. Thus, the enzymatic treatment of the method of the present invention initiates and accelerates hydrolysis prior to biodegradation by microbial action. That is, if the treatment method of the present invention is not used, the target copolyester remaining in the general waste context will not be biodegraded effectively or will biodegrade more slowly. This is desirable for composting, where hydrolysis performed solely by microbial action degrades the polymer backbone of the aromatic-aliphatic copolyester into low molecular weight compounds that can be absorbed and catabolized by microorganisms. Because it is relatively slow, or at least slower.

  Enzymatic treatment is performed by contacting a hydrolytic enzyme with the high aromatic content copolyester. Contact includes treatment of any type of copolyester with a hydrolase, including but not limited to spraying, painting, coating, pouring, mixing, applying, and the like. Contact can be made during production and use of the copolyester and after disposal of the copolyester. That is, the present invention hydrolyzes during manufacture or use into products containing high aromatic content copolyesters that do not retain consumables or come into contact with skin or mucous membranes to initiate and accelerate the biodegradation process. It is contemplated to apply the enzyme. The terms “pre-treatment”, “post-treatment” and “treatment” are synonymous and each means contacting the subject copolyester with a hydrolase. The meaning of these terms is not necessarily defined by when the hydrolase is applied to the subject copolyester. For example, contacting hydrolytic enzymes before use or disposal is considered “pretreatment” or “treatment”. Similarly, hydrolase contact at any point after use or after disposal is considered “post-treatment” or “treatment”.

  The specific content of contacting the subject copolyester with the subject hydrolase is indeterminate and depends on the nature of the disposable product and the expected disposal route. In general, with respect to composting and landfill waste contexts, and solid waste, grit and sludge recovered from wastewater treatment systems, there are various embodiments in which the subject copolyesters are contacted with hydrolytic enzymes. After waste collection, including collection of recyclable items, solid waste that is not recyclable is separated from recyclable waste. A non-recyclable solid waste containing an aqueous solution containing from about 0.1 to about 10.0% by weight of at least one hydrolase or hydrolase mixture so that the aqueous solution contacts the target copolyester. Can be applied. Such contact may include spraying on solid waste. A second embodiment of contact involves coating an article containing the subject copolyester. As an alternative, such articles are painted with or immersed in an aqueous solution of hydrolase. These examples are not intended to limit the present invention; the present invention contemplates virtually any method of contacting an aqueous solution containing a hydrolase with a subject copolyester. Further, depending on the nature of the disposable article and the waste context, it is contemplated that the hydrolytic enzyme is applied multiple times to contact the solid waste.

  For example, for waste discarded in municipal composting facilities, apply an aqueous solution of at least one hydrolase enzyme at any point during the waste recovery process and after the waste arrives at the composting facility. can do. Since composting takes place under conditions of high humidity and temperature, it is contemplated that the biodegradation achieved by microbial action is appropriately accelerated by applying the hydrolase solution several times or even once. While it is important to directly contact an article containing a highly aromatic copolyester with a hydrolase, a waste mass containing a higher percentage of such article is added to the solution containing the hydrolase. Fully saturate. As an alternative. Thorough spraying is sufficient for the amount of waste containing a small amount of such articles.

  Until the cell is full and closed, the waste is sorted daily from solid waste and / or wastewater treatment in landfills due to the landfill properties, which are placed in the same cell, hardened and obscured. The contact of the hydrolytic enzyme with the solid waste is generally performed after the waste is dumped in the landfill. It is contemplated to apply the hydrolase solution multiple times to the cell until it is sealed. Traditional “dry trash grave” landfills do not achieve composting temperature and humidity, but can achieve higher temperatures because they are sealed and because they are generally anaerobic and methane-producing: microorganisms Hydrolysis due to water occurs more slowly than in composting contexts, and landfill degradation takes decades compared to months or years in compost. Nevertheless, contacting the hydrolytic enzyme with the copolyester of interest in conventional landfills will accelerate even if biodegradation occurs there. In bioreactor landfills, the degradation conditions are close to those of composting due to the increased humidity and air content. Due to the more advantageous conditions, the application of hydrolase to the waste context and the highly aromatic copolyester in the waste has the effect of accelerating the biodegradation rate faster than in dry grave burial sites.

  When the method of the present invention is useful, there are at least two stages in the wastewater treatment process: In the initial sorting of solid waste, at any point after sorting and before dumping the waste in the landfill Then, the hydrolase solution is brought into contact with the solid waste; later, when the sludge and scum enter the digester, the hydrolase solution is added to the digester to promote the degradation of the copolyester particles. And, of course, after the grit and / or digested material is dumped into the landfill, the hydrolytic enzyme can be contacted.

Simulating composting in an Aqua bioreactor Composting is a heterogeneous complex environment containing complex organic materials and inorganic particulate matter. Matching operating conditions from one experiment to another is difficult to maintain. Furthermore, standard analytical methods cannot be easily applied. Therefore, a water bioreactor was designed to evaluate the effect of hydrolase treatment on aliphatic-aromatic copolyesters. The use of an aqueous reaction system that characterizes the biodegradability / compostability of the polymer has been reported previously and facilitates control of experimental variables. See Kleeberg et al., Supra; Gooda et al., Supra; and German testing standard DIN V54900, Deutsches Institute for Normburg 1998 Testing of the com- parsity of polymers. In the method of the present invention, the conditions in the water bioreactor were very similar to the conditions found in the composting environment (55-60 ° C., 100% relative humidity). The data obtained from the water bioreactor experiments herein is similar to that expected from a municipal composting system. Ultimately, the determination that waste biodegradation is accelerating relies on measuring the amount of solid polymer remaining in the bioreactor after enzyme treatment.

Bioreactor Bioreactor # 1 was a 500 mL standard jacketed glass container with a screw cap (Bellco Glass, Stock # 1965-50500 W / J). The bioreactor had an electromagnetic stirrer to provide continuous stirring and was attached to the controller to maintain the pH at 8.0. In order to keep the temperature inside the bioreactor constant, water was passed through the bioreactor jacket using a circulating water bath. Air was bubbled through the bioreactor medium at a flow rate of 50 mL / min.

Compost tea Compost tea was prepared as reported in the literature with minor modifications. In a separate 500 mL screw-cap glass Erlenmeyer flask, about 9 g of each compost was added to 100 mL of SMV1 medium. The flask was incubated for 16 hours at 55 ° C. in a shaking water bath. The upper portion of the liquid in each flask (50 mL) was poured into a separate 50 mL centrifuge tube. The tube was left for 5 minutes to allow the largest particles to settle. The top 40 mL of liquid was removed from each tube and placed in a new 50 mL centrifuge tube. The tube was centrifuged at 5000 rpm for 5 minutes. Each pellet was resuspended in 10 mL of SMV1 medium. All the resuspended pellets were combined to form a concentrated compost tea.

Degradation Measurements Many methods for measuring degradation of polymers are known in the art. Examples of these include physical observation, gravimetric analysis, biogas (ie carbon dioxide) production, biomass accumulation, degradation product characterization (eg using standard analytical techniques such as GC, LC, MS, UV, etc.) ), And particle size analysis. Specifically, the degradation rate per unit time can be determined using the amount of solid polymer measured before and after the enzyme treatment. In addition, a sample can be taken and an optical particle size analyzer can be used to measure the relative particle size and concentration of the polymer particles over time according to various conditions. The ability of one or more microorganisms to use a polymer as a carbon source can be measured by an increase in total biomass.

Culture system The classical batch culture method is a closed system where the composition of the medium is set at the beginning of the culture and is not artificially altered during the culture process. Thus, at the beginning of the culturing process, the medium is inoculated with the desired organism and nothing is added to the system to produce growth or metabolic activity. However, in general, “batch” cultures are batches for the addition of a carbon source, and attempts are often made to control factors such as pH and oxygen concentration. In a batch system, the metabolite and biomass composition of the system constantly change until the end of the culture. Within a batch culture, the cells enter a high growth log phase via a static induction phase and eventually transition to a stationary phase where the growth rate decreases or stops. If untreated, the stationary phase cells eventually die. Log phase cells are often responsible for the size (bulk) of final product or intermediate production in some systems. Generation after stationary or logarithmic phase can be obtained in other systems.

  A variation of the standard batch system is a semi-batch system. A semi-batch culture process is also suitable for the present invention and includes a general batch system, except that the substrate is added in incremental increments as the culture progresses. Semi-batch systems are useful when suppression of catabolic metabolites tends to inhibit cellular metabolism, and when it is desirable to have a limited amount of substrate in the medium. Measuring the actual substrate concentration in a semi-batch system is difficult and is therefore estimated based on changes in measurable factors such as pH, partial pressure of waste gases such as dissolved oxygen and carbon dioxide. Batch and semi-batch culture methods are well known in the art and examples include Thomas D. et al. Block, In Biotechnology: A Textbook of Industrial Microbiology (1989) 2nd Ed. Sinauer Associates, Inc. , Sunderland, MA ("Block") or Deshande, Mukund V. (1992) Appl. Biochem. Biotechnol. 36: 227-234.

  Commercial production can also be achieved with continuous culture. Continuous culture is an open system in which a defined medium is continuously added to the bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. In continuous culture, cells are generally maintained at a constant high liquid phase density, at which the cells are primarily in log phase growth. As an alternative, continuous culture can be performed on immobilized cells where carbon and nutrients are added continuously and useful products, by-products or waste are continuously removed from the cell mass. . Cell immobilization can be performed using a wide range of solid supports composed of natural and / or synthetic materials.

  Continuous or semi-continuous culture allows one factor or any number of factors that affect cell growth or end product concentration to be modulated. For example, in one method, limiting nutrients such as carbon sources or nitrogen levels can be maintained at a fixed rate and all other parameters can be adjusted. In other systems, many factors that affect growth can be varied continuously while maintaining a constant cell concentration as measured by medium turbidity. Continuous systems attempt to maintain steady state growth conditions, and therefore cell loss due to media being removed must be balanced against cell growth rate in culture. Methods for regulating nutrients and growth factors in continuous culture processes and techniques for maximizing product formation are well known in the field of industrial microbiology, and various methods are detailed by Block, supra. ing.

  The invention is further defined in the following examples. It should be understood that these examples are given by way of illustration only. From the above description and these examples, those skilled in the art can ascertain the essential characteristics and to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. Various changes and modifications can be made to the present invention.

General Methods Materials and methods suitable for maintaining and growing bacterial cultures are well known in the art. A technique suitable for use in the following examples is described by Gerhardt, P .; Et al., Eds. , MANUAL OF METHODS FOR GENERAL BACTERIOLOGY (1994), American Society for Bacteriology, Washington, DC, or Block (above). Unless otherwise specified, all reagents and materials used for the growth and maintenance of bacterial cells are Aldrich Chemicals (Milwaukee, WI), DIFCO Laboratories (Detroit, MI), GIBCO / BRL (Gaithersburg, MD). Or from Sigma Chemical (St. Louis, MO).

  The hydrolase, or lipase, used in the following examples is a product of Novozymes (Denmark), commercially available from Sigma Chemical. Novozyme ™ 871 is a lipase derived from Thermomyces lanuginosus (CAS 9001-62-1; EC 3.1.1.5; Sigma product number L0902). Palatase 20000L is a lipase derived from Rhizomucor miehei (CAS 9001-62-1; EC 3.1.1.5; Sigma product number L4277). Lipolase 100L is a lipase derived from Thermomyces lanuginosus (CAS 9001-62-1; EC 3.1.1.5; Sigma product number L0777).

  The polymers disclosed in the present invention can be synthesized by those skilled in the art or purchased from Aldrich Chemicals. The polymer tested for degradation has the following composition: “Polymer A” is 17.5 mol% DBE-5 (dimethyl glutarate, CAS # 1119-40-0, based on 100 mol% total acid), DRL-6 is a sulfonated aliphatic-aromatic polymer comprising poly (ethylene terephthalate) with 2 mol% (dimethyl 5-sulfoisophthalate, sodium salt, CAS # 3965-55-7); “Polymer B” is , DBE-5 17.5 mol% (dimethyl glutarate, CAS # 1119-40-0, based on 100 mol% total acid), DRL-6 2 mol% (dimethyl 5-sulfoisophthalate, sodium salt, CAS # 3965-55-7), and poly (ethylene glycol) 8 wt% (1000 MW, bop) together with poly ( Sulfonated aliphatic consisting Chi terephthalate) - is an aromatic copolyesters.

  The meanings of the abbreviations are as follows: “h” is hours, “min” is minutes, “sec” is seconds, “d” is days, “mL” is milliliters, “L” is liters, and “μL” is Microliters, “g” means grams, “mg” means milligrams, “μg” means micrograms, “M” means moles, “mM” means millimoles, “μM” means micromoles, “OD” = designated wavelength The optical density at.

Example 1
Enzymatic assay to determine the effect of lipase on various substrates This example describes how treatment with lipase releases terephthalic acid from an aliphatic-aromatic copolyester. The polymer substrate was ground in a Bantam Micropulverizer (Mikropul, Summit, NJ) using a final sieve size of 0.006 inches. Samples of the polymer substrate were incubated with lipase (Novozyme ™ 871) in a 125 mL screw-top glass Erlenmeyer flask. The polymer substrate was suspended in 10 mM potassium phosphate buffer (pH 7.0) at a final concentration of 0.4% (w / v). Enzyme was added from the stock solution (0.5 units / mL) to the flask to a final concentration of 10 units / mL. Control flasks with buffer only, enzyme only, and substrate only were also prepared. The flask was closed tightly and incubated at 55 ° C. in a shaking water bath. Samples were withdrawn at 24 hours (T ₂₄ ) and 48 hours (T ₄₈ ) after the initial sample (T ₀ ). The sample was passed through a 0.45 micron Acrodisc® syringe filter (Pall, Ann Arbor, MI). The amount of terephthalic acid released from each polymer substrate during the incubation was determined for each sample by measuring UV absorbance at λ = 240 nanometers (Abs ₂₄₀ ). Abs ₂₄₀ values were averaged at each sample time for duplicate flasks. By subtracting the T ₄₈ Mean Abs ₂₄₀ for the polymer which does not use an enzyme from T ₄₈ Mean Abs ₂₄₀ of the same polymer using enzymes, absorbance caused by the enzyme activity after 48 hours (i.e., released terephthalate Change).

The Abs ₂₄₀ value of the substrate incubated with the enzyme was consistently higher than the Abs ₂₄₀ value of the same substrate incubated without enzyme. The ΔAbs ₂₄₀ at the T ₄₈ value indicates that the maximum amount of terephthalic acid was released from polymer B and the minimum amount of terephthalic acid was released from PET.

^a ２つ組のフラスコからの試料の値の平均。
^b ΔAbs₂₄₀ = (酵素を有する基質YのAbs₂₄₀) - (基質YのAbs₂₄₀) Table 1. Release of terephthalic acid from various sample polymers treated with lipase Novozyme ^TM 871

^a Average of sample values from duplicate flasks.
^b ΔAbs ₂₄₀ = (Abs _{240 of} substrate Y with enzyme)-(Abs _{240 of} substrate Y)

Example 2
Enhanced degradation of lipase-treated polymer B In this example, how to degrade polymer more quickly by microbial populations derived from compost by first treating polymer B with a commercial lipase. Describe what will be possible.

Initial treatment of polymer B with lipase and subsequent treatment of polymer B with microbial aggregates were performed in bioreactor # 1. Bioreactor # 1 was a 500 mL standard jacketed glass container with a screw cap (Bellco Glass, Stock # 1965-50500 W / J). The bioreactor had an electromagnetic stirrer to provide continuous stirring and was attached to the controller to maintain the pH at 8.0. To keep the temperature inside the bioreactor constant (initially 55 ° C., 58 ° C. after 42 days of operation), a circulating water bath was used to pass water through the bioreactor jacket. Air was bubbled through the bioreactor medium at a flow rate of 50 mL / min. The bioreactor initially was 350 mL of SMV1 medium (0.05 M potassium phosphate buffer (pH 8.0), 0.01 M ammonium sulfate, 0.001% yeast extract, 2 mM MgCl ₂ , 0.7 mM CaCl ₂ , 0.05 mM MnCl _2. , 0.001 mM ZnCl ₃ , 0.002 mM thiamine hydrochloride, 1.72 μM CuSO ₄ , 2.53 μM CoCl ₂ , 2.42 μM NaMoO ₂ , 0.005 g vitamin B12, 0.005 g p-aminobenzoic acid, and BME vitamin 0.5 mL / L (Sigma-Aldrich Chemical)) was included with 0.4% (w / v) polymer B. Commercially available lipases Novozyme 871 (final concentration 10 units / mL), Palatase 20000L (final concentration 10 units / mL) and Lipolase 100L (final concentration 1 unit / mL) were added to the bioreactor. Another bioreactor, essentially the same as bioreactor # 1, was operated simultaneously as a control to evaluate the effectiveness of lipase treatment. Bioreactor # 2 contained SMV1 medium with 0.4% PET and the same commercial lipase as bioreactor # 1. Bioreactor # 3 contained SMV1 medium with 0.4% PET without the addition of lipase. Bioreactor # 4 contained SMV1 medium with 0.4% polymer B without the addition of lipase. Lipase was added to bioreactor # 1 and bioreactor # 2 at the beginning of the experiment (day 1) and on day 15.

  Microorganisms as concentrated “compost tea” produced in 4 different composts (2 different composts: crab shell and sawdust compost, poultry litter compost, and cut grass garden) 24 hours after operation All of the bioreactors were incubated with. In a separate 500 mL screw-cap glass Erlenmeyer flask, about 9 g of each compost was added to 100 mL of SMV1 medium. The flask was incubated for 16 hours at 55 ° C. in a shaking water bath. The upper portion of the liquid in each flask (50 mL) was poured into a separate 50 mL centrifuge tube. The tube was left for 5 minutes to allow the largest particles to settle. The top 40 mL of liquid was removed from each tube and placed in a new 50 mL centrifuge tube. The tube was centrifuged for 5 minutes at 5000 rpm in a Sorval T6000D tabletop centrifuge. Each pellet was resuspended in 10 mL of SMV1 medium. All the resuspended pellets were combined to form a concentrated compost tea. Each bioreactor was incubated with 3.5 mL of concentrated compost tea. A sample (5 mL) for particle size analysis was removed from the bioreactor while the medium was continuously stirred. Samples were mixed with an equal volume of ethanol and analyzed on the same day with a laser diffractometer (Beckman Coulter LS230).

  The particle size distributions of polymer B and PET particles were plotted in a volume weighted distribution, highlighting the presence of large particles, especially particles greater than 40 μm in diameter. There was no significant difference in particle size distribution for the initial samples from either bioreactor (data not shown). On day 90, the particle size distribution of the lipase-treated polymer B was significantly different from the untreated polymer B (FIG. 1). Untreated polymer B has a broad particle size distribution ranging from less than 10 μm to about 600 μm, with the majority of particles ranging from 200 μm to 600 μm, whereas lipase-treated polymer B has this particle size Lack of particles in the range. Furthermore, the peak particle size of lipase-treated polymer B (about 150 μm) was much lower than the peak particle size of untreated polymer B (about 250 μm). These data indicate that treatment with lipase significantly promotes polymer B biodegradation by the microorganisms initially contained in the concentrated compost. On the other hand, treatment with lipase and incubation with microorganisms contained in concentrated compost tea had little or no effect on PET (FIG. 2).

Figure 6 shows the effect of lipase treatment on the degradation of polymer B in an aqueous reactor after enzyme treatment. Figure 3 shows the effect of lipase treatment on the degradation of polyethylene terephthalate (PET) as measured by a decrease in polymer particle size distribution in an aquatic bioreactor.

Claims (29)

A method for increasing the hydrolysis of aliphatic-aromatic copolyesters by microorganisms, comprising:
The copolyester is
At least one aromatic dicarboxylic acid or ester thereof, and at least one aliphatic dicarboxylic acid or ester thereof, wherein the aromatic acid is more than 60 mol% of the total dicarboxylic acid in the copolyester and about 99 mol% Occupy up to
Contacting the copolyester with at least one hydrolase in aqueous solution.   The method of claim 1, wherein the copolyester comprises at least about 61 mol% to about 90 mol% of at least one aromatic dicarboxylic acid or ester, based on the total dicarboxylic acid in the copolyester.   The method of claim 2, wherein the copolyester comprises at least about 70 mol% of at least one aromatic dicarboxylic acid or ester, based on the total dicarboxylic acid in the copolyester.   The method of claim 2, wherein the copolyester comprises at least about 75 mol% of at least one aromatic dicarboxylic acid or ester, based on the total dicarboxylic acid in the copolyester.   The method of claim 2, wherein the copolyester comprises at least about 80 mol% of at least one aromatic dicarboxylic acid or ester, based on the total dicarboxylic acid in the copolyester.   The at least one aromatic dicarboxylic acid or ester is terephthalic acid, dimethyl terephthalate, isophthalic acid, dimethyl isophthalate, 2,6-naphthalenedicarboxylic acid, dimethyl-2,6-dicarboxylic acid, dimethyl-2,6-naphthalene. And the at least one aliphatic dicarboxylic acid or ester is selected from the group consisting of succinic acid, dimethyl succinate, glutaric acid, dimethyl glutarate, 2-methylglutaric acid, 3-methylglutaric acid, 2. The method of claim 1 selected from the group consisting of adipic acid, dimethyl adipate, oxalic acid, dimethyl oxalate, malonic acid, dimethyl malonate, and mixtures thereof.   The method of claim 6, wherein the copolyester comprises from about 1 mol% to less than about 40 mol% of at least one aliphatic acid or ester, based on the total dicarboxylic acid in the copolyester.   The method of claim 6, wherein the copolyester comprises from about 20 mol% to less than about 40 mol% of at least one aliphatic acid or ester, based on the total dicarboxylic acid in the copolyester.   The method of claim 1, wherein the copolyester is combined with starch, protein, cellulose, wax, filler, silicate filler, polylactic acid, polyhydroxyamide, or mixtures thereof.   The method of claim 3, wherein the copolyester further comprises at least one diol.   The at least one diol is 1,2-ethanediol, 1,3-propanediol, 1,4-butanediol, 1,6-hexanediol, ethylene glycol, di (ethylene glycol), tri (ethylene glycol) 11. The method of claim 10, wherein the method is selected from the group consisting of: poly (ethylene glycol), poly (alkylene ether) glycol, poly (propylene ether) glycol, and mixtures thereof.   The method of claim 1, wherein the copolyester further comprises a sulfonated compound.   13. The method of claim 12, wherein the copolyester comprises from about 0.1 to about 10.0 mole percent of a sulfonated compound, based on the total diol or total dicarboxylic acid in the copolyester.   13. A method according to claim 12, wherein the sulfonated compound is selected from the group consisting of sulfonated mono- or dicarboxylic acids, or esters or metal salts thereof.   The sulfonated compound is selected from the group consisting of sulfosuccinic acid, 3-sulfobenzoic acid, 4-sulfobenzoic acid, 5-sulfosalicylic acid, sulfophthalic acid, sulfoterephthalic acid, and 5-sulfoisophthalic acid, or esters or salts thereof. 13. The method of claim 12, wherein:   The method of claim 12, wherein the copolyester comprises from about 20 mol% to less than about 40 mol% of at least one aliphatic carboxylic acid or ester, based on the total dicarboxylic acid in the copolyester.   The method of claim 12, wherein the copolyester further comprises at least one diol.   The method of claim 1, wherein the copolyester comprises about 17.5 mol% dimethyl glutarate, about 2 mol% dimethyl 5-sulfoisophthalate, sodium salt, based on the total dicarboxylic acid in the copolyester.   The method of claim 18, wherein the copolyester further comprises about 8 wt% poly (ethylene glycol).   The method of claim 1, wherein the aqueous solution comprises about 0.1 to about 10% by weight of at least one hydrolase.   The method of claim 1, wherein the at least one hydrolase is selected from the group consisting of proteases, lipases, cutinases, esterases, and combinations thereof.   The method of claim 6, wherein the at least one hydrolase comprises a lipase.   The method of claim 10, wherein the at least one hydrolase comprises a lipase.   The method of claim 12, wherein the at least one hydrolase comprises a lipase.   The contact of the at least one hydrolase is selected from the group consisting of spraying, painting, coating, applying, treating, and mixing the at least one hydrolase and the copolyester. The method according to 1.   The method of claim 1, wherein the copolyester is contacted with the at least one hydrolase prior to degradation of the copolyester by the microorganism.   The method of claim 1, wherein the copolyester is located in a waste context.   28. The method of claim 27, wherein the waste context is a solid waste, compost, or wastewater treatment system.   The contact of the at least one hydrolase is selected from the group consisting of spraying, painting, coating, applying, treating, and mixing the at least one hydrolase and the copolyester. 28. The method according to 27.

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