Journal of Scientific & Industrial Research Vol.59, June 2000, pp 433-445

Bioplastics

V C Kalia*, Neena Raizada and V Sonakya Centre for Biochemical Technology, Mall Road, University Campus, Delhi II 0 007, India

Plastics and polymers have become a part of our life today. The annual consumption of plastics in India is 2 kg/person/y. In the other developed countries the per capita consumption of plastics is of the order of 60 kg/y. Some naturally occurring plastic materials have been made use of by man from the earliest times, e.g., Lac and Amber. Today, almost all the pl astics are manufactured synthetically and they have much better properties than naturally occurring plastics. The basic raw material for all the modern plastics is crude oil and natural gas . Due to their non-biodegradable nature, environmentalists are campaigning against their production and usage. In India, pl astic wastes accounts for I to 4 per cent by weight of the total of 80,000 metric tonnes of Municipal solid waste generated daily. In USA, of the 4 lakh tonnes of garbage generated everyday, plastic constitute 30 per cent of its volume. Pl astics (polymers) can be degraded by three different processes, either independently or in combination : (i) Light or high energy radi ation, (ii ) Heat, and (i ii ) Microbes. In response to increasing public concern over environmental hazard caused by plastic, many countries are conducting various solid waste mauagement programmes including plastic waste reduction by development of biodegradabl e plastic materi al. There is an intense research fo r making the biodegradable plastic material. Some biodegradable pl astic materials under development are: (i) Poly hydroxy alkanoates (PH As) , (ii) Poly-lactides. (i ii ) Aliphatic poly-esters, (iv) Polysaccharides, and (v) Co-polymers and/or blends of above. However, it is possible to produce biodegradable plastics from bio-wastes with the help of a syntrophi c population of microbes of diverse origins. Thi s will help to reduce waste management problems and improve environ-ment.

Plastics and polymers have become a part of our life today. The annual consumption of plastics in India is 2 kg/person/y. When compared with the rest of the world this is a very small figure. An average American uses 80 to 90 kg of plastics/y. In other developed countries the per capita consumption of plastics is of order of 60 kg/y and the world average is 15 kg/y. The projected demand of plastics in India is about 25 lakh tonnes/y by the end of this century. There were about 12,500 plastic process-ing units in India with a total investment of Rs 2,000 crores in 1992. By 2000, we would need about 20,000 processing units which would require an additional in-vestment of Rs 3,000 crores . In the Indian context, it must be borne in mind that the growth ofthe Indian plas-tics industry has been phenomenal, where the growth rate is higher than the plastics industry elsewhere in the world 1• From 1.88 million tonnes ( 1995-96) the domes-tic demand for plastics is expected to cross 4 million tonnes by year 200 l-2. Furthermore the depletion of al-ready scarce natural resources has establi shed plastics as the material of choice in innumerable applications. For instance, in packaging, plastics are increasingly re-

*Author for correspondence Phone: 091-011- 725 72 98; 725 61 56, Fax:09 1-011 - 7257 471 & 741 64 89, e- mai l: vckali a@cbt.res.in

placing jute, paper, wood, glass and metal. Even tradi-tional areas, such as agriculture, are also using plastics as part of modern scientific methods for packaging.

Some naturally occurring plastic materials have been made use of by man from the earliest times . Lac was used for molded ornaments and Amber, a yellow coloured foss il polymeric resin formed from trees buried into the ground millions of years ago, was known to the Greeks2•

Synthetic Plastics Today, almost all the available plastics are manufac-

tured synthetically and they have much better properties than naturally occurring plastics. The basic raw mate-rial for all the modern plastics is crude oil and natural gas.

Crude oil is recovered from underground oil deposits or from the seabed by offshore drilling. This oil is a mix-ture of heavy hydrocarbons and is processed in the pe-troleum refineries to obtain a vide spectrum of industry. Heavy oil molecules when heated under pressure and in the presence of a catalyst breakdown into smaller mol-ecules. This process is called catalytic cracking3. The resultant mixture of hydrocarbons is then subjected to fractional di stillation . The fraction s evaporated at dif-ferent temperatures can be condensed separately. Naph-tha is one such fraction. Naphtha, which closely re-

434 J SCI IND RES VOL 59 JUNE 2000

sembles the petrol used in automobiles, is the main source of chemicals used for making various kinds of plastics. Naphtha is further cracked to obtain still lighter hydro-carbons, which are the raw materials for most of the com-mon plastics used today. Polyethylene and PVC are the two major plastics, manufactured from ethylene gas ob-tained by cracking naphtha. Natural gas, which also comes out from the oil-fields is a source of yet another important raw material for plastics, called formaldehyde. This chemical which is the raw material for making rigid types of plastics is made from methanol produced from natural gas4 . There are many other chemicals, which also serve as starting materials for various types of polymers and plastics. Almost all of them are derived from crude oil. Butylene is extensively used for making synthetic rubber.

Plastic vs Pollution The production and consumption of plastics have in-

creased in geometrical progression in this century. How-ever, due to their non-biodegradable nature, environmen-talists are campaigning against their production and us-age. In India, plastic wastes accounts for I to 4 per cent by weight of the total of 80,000 metric tonnes of Mu-nicipal solid waste generated daily'. In USA, of the 4 lakh tonnes of garbage is generated everyday, where plas-tic constitute 30 per cent of its volume3.

Every one sees plastic waste dumps daily, but no con-certed effort is evident for their disposal. Plastic waste is managed largely by the unorganized section of our society- the rag pickers . These wastes are recycled for producing low quality plastic products. Only I 0 per cent of the disposed plastic is recycled through this route. The remaining non-recovered and unused waste takes a very long time to degrade and be reduced to dust. The plastic waste pose a serious problem to landscape, wild-life, marine animals, and become an "environmental eye sore". It is primarily due to the non-biodegradable na-ture of the plastics. Plastics waste management status in India5 is given in Table l.

Plastics have also a costly impact on waste manage-ment and municipalities are becoming aware of the sig-nificant savings that collection of 'wet' organic wastes in so-called 'biobins' to be composted can provide. For these reasons, reaching the conditions for replacement on non-degradable polymers by degradable plastics, par-ticularly for single-use disposable and packaging appli-cations, is of major interest both to decision-makers and the plastic industry0 •

Table I -Plastics waste management status in lndia5

Consumption of plasti cs Waste available for recycling Total

Anti-plastic Drive

1995-96 Est imates by 200 I (Thousand tonnes)

1889 800 2689

4374 2000 6374

Many Indian states have issued notifications for the use of cert"ain types of plastics, particularly for packing food stuff. Government has issued notification to ban coloured plastic bags. UP government has also planned to ban poly-bags soon. Similarly, Himachal Pradesh gov-ernment has introduced the HP Non-biodegradable Gar-bage (Control) Act 1995. It prohibits throwing or depos-iting plastic articles in public places and facilitates col-lection through garbage in identifiable and marked gar-bage receptacles for non-biodegradable. In a step towards eradication of plastics in daily use, Sabarimala, Kerala's major pilgrim shrine, and the state zoos and museums have banned carrying of plastic bags and containers on their premises .

Ministry of Environment and Forest have issued cri-teria for labeling "plastic products" as "Environmental Friendly" under its 'Ecomark' scheme in association with the Bureau of Indian Standards . One of the requirements for plastic products, is that the material used for packag-ing shall be recyclable or biodegradable . Among the various guidelines, a clause reads "no recycled plastic waste shall be used" for critical applications like plastic piping system, water-storage tanks, packaging for food articles5.

The Government of India has issued the new guide-lines under the National Plastic Waste Management Task Force's (NPWMTF) for plastic industries I manufactures. The salient features of the NPWMTF's report are 3:

(a) The strategy for effective management of plastics wastes should entail the three Rs: Reduce, Reuse and Recycle;

(b) Manufacture of dirty coloured carry bags with vis-ible contamination and their circulation in the mar-ket should be banned;

(c) Thickness of plastic bags to increase from 6 11 to 20 11 for virgin plastic bags and 25 mu for those made out of recycled plastics; and

Method

Photo-degradation

Thermo-oxidative degradation

Bio-degradation

KALlA et al.: BIOPLASTICS

Table 2- Polymer degradation pathways

Active Heat Degradation Eco- Acceptability agent required rate friendly

UVor No Slow initiation No Yes, other but fast expensive high energy propogation radiations

Heat +02 Very high Fast No No

Microbial No Moderate Yes Yes, highly enzymes acceptable

·~ Light ) ~+~+~ Degradable Products Photo degradable Polymers High Energy

Microbes ....-..6--D ~ + ...,..A...tJ

Degradable Products

Microbes --~---- ~ ------

Degradable blend

Photosensitive linkage

Microbial sensitive linkage

Non Biodegradable polymers

0 Biodegradable polymers in blend Figure I -Photo and biodegradation of polymers and blends

Plastic Degradation

435

(d) Virgin bags to be colourless, i.e., translucent, while recycled bags to be in colour.

This never ending waste generation will lead to accu-mulation of plastic in our environment. There is thus need to develop a eco-friendly strategy for up-gradation of recycling technologies, re-defining guidelines for dis-posal, formulating specifications and codes of practices (Regulations and Legislation) .

Polymers can be degraded by three different processes given in Table 2, either independently or in combina-tion: (i) Light or high energy radiation, (ii) Heat, and (iii) Microbes7 (Figure 1).

(i) Photo Degradation - It is initiated by the absorp-tion of light energy by reactive groups of the poly-mer, leading to smaller fragments. The process re-

436 1 SCI IND RES VOL 59 JUNE 2000

quires either in-built photo-responsive group or an additive.

(ii) Heat- Degradation of a polymer occurs by heat-ing in the presence of oxygen, i.e., thermo-oxidative degradation. At the primary stage, macro-molecules bonds are broken down with the formation of radical sites. These radical sites react with oxygen to form per-oxy radicals. Thus, long chain polymer molecules are converted into smaller fragments and volatiles.

(iii) Biodegradation - For following the biological route, polymers have to be designed in a manner that monomers are susceptible to microbial attack. Alter-natively, biodegradable additives or groups have to be blended during polymer formation.

Biological transformation occurs via relatively spe-cific enzymatic processes. Microbes have a significant and dominant role to play. The process is favoured by darkness, high humidity, adequate minerals and other nutrients, temperature, pH, and 0 2• Biodegradation is a gradual and slow decomposition of complex materials by living micro-organisms, e.g., fungi, bacteria, and ac-tinomycetes. Aerobic degradation of complex organic material works efficiently till 60 per cent. On the other hand, anaerobic degradation of organic matter to C02 and CH

4 occurs at a very high rate of up to 95 per cent.

The rest of the matter being inorganic materials easily mixes with soil and increases soil quality. Fungi have been more employed because of their evident invasion properties.

Hydrolysis Hydrolysed Oxidation

POLYMER Product

(Microbial)

C02 + Hp (Microbial)

Microbial Biodegradation of Polymers

Biological Approach for pollution Abatement In response to increasing public concern over envi-

ronmental hazard caused by plastic, many countries are conducting various solid waste management programmes including plastic waste reduction by development of bio-degradable plastic materialx. Thus, there is an intense research going on for making the biodegradable plastic material. Some biodegradable plastic materials under de-velopment are: (i) Polyhydroxy alkanoates (PHAs), (ii) Polylactides, (iii) Aliphatic polyesters, (iv) Polysaccha-rides, and (v) Co-polymers and/or blends of above.

The target can be achieved by either producing plas-

tics containing biological additives2 (e.g. starch) or pro-duce plastics from biological materials exclusively. The introduction of additives like starch into polyethylene-"Bio-D" bags help it to undergo biodegradation. Starch acts as nutrition for microbes. The major limitation of this type of plastic is the quantity of starch, which can be used as additive, cannot exceed 30 per cent. The degradation rate of this photo-oxidant is quite slow.

A eco-friendly alternative to this "biodegradable poly-mer" is the production of poly-hydroxy butyrate (PHB). PHB is 100 per cent biodegradable plasticx. It can be prepared from renewable sources. Biopol is a PHB which degrades within few months in the soil. However, it is very costly, roughly 25-times costlier than polyethylene, and at present its use is restricted to medical applica-tions only, such as nails for bone consolidation, suture thread, and pharmaceutical capsules. These bio-plastics can potentially replace traditional plastics or petroleum based plastics.

Potential microbial routes to the production of plas-tics and synthetic fibres can be divided mainly into three categories. These are: (i) The production of bio-poly-mers processing the properties of plastics, (ii) The use of microorganisms as bio-catalysts to produce specialised chemical intermediates or monomers, and (i ii) The re-lease of usable chemicals from renewable carbon sources such as lignin9• Microorganisms are being used to produce environmentally-benign plastics.

Polyhydroxy alkanoates (PHAs) are biopolyesters accumulated as storage compounds by a large number of different prokaryotes. More than 100 different hy-droxy-alkanoic acids have already been detected as con-stituents of these bacterial polyesters. They are biode-gradable thermoplastics and elastomers which have at-

. tracted considerable attention from academia and indus-try for their various technical applications in industry, agriculture, medicine, pharmacology and other areasH>.

PHA is a biodegradable polymer material that accu-mulates in numerous microorganisms under unbalanced growth conditions and has been produced on an indus-trial scale by ZENECA by using Alcaligens eutrophus. PHA is synthesized and accumulated as granules in the cytoplasm of bacterial cells . The native PHA granules that contain lipids and proteins are rapidly hydrolyzed by intracellular de-polymerases under appropriate con-ditions. High resolution 13C NMR spectroscopy of whole cells containing poly-3-hydroxybutyrate (PHB) has re-vealed that PHB is present predominantly in a higher mobile amorphous state, which seems to allow easy ac-

KALlA et al.: BIOPLASTICS 437

cess of de-polymerases. Freezing and storage of the PHA granules at low temperature, treatment with acetone or a hypochlorite solution, or even application of strong centrifugal forces to cells containing PHA could trigger irreversible conversion ofPHA to the crystalline state 11 • Recently, transmission electron microscope examination of freeze-dried PHA granules revealed that the dry gran-ules have a non-crystalline core and a crystalline shell morphology.

Poly-hydroxy alkanoates (PHAs) are microbial poly-esters which have received considerable attention as biodegradable alternatives to conventional plastics . Co-polymers of hydroxy-butyrates and hydroxy-valerate including poly (~-hydroxybutyrate-co-~-hydroxy-valerate) (PHBV), from plastics having good mechanical qualities and have been made to incorporate low-value materials into PHB V s to reduce their overall cost. Com-starch is a particularly attractive filler for PHBV-based composites. Moreover, methods for blending starch with several polymers to produce blown film and extruded composites have been developed. Significant biodegra-dation occurred only after colonization of the plastic, a parameter that was dependent on the resident microbial populations 12 •

Intracellular energy reserves , such as poly-3-hydroxybutyrate (PHB) may also promote environmen-tal persistence. PHB is a homo-polymer of 3-hydroxy-butyric acid, which some bacteria accumulate during unbalanced growth to provide an endogenous source of carbon and energy. Indirect evidence for the occurrence of PHB in Legionella pneumophila has been provided by Fourier transform infrared spectroscopy of whole cells and by pyrolysis mass spectrometry (MS). Legionella species metabolize 3-hydrobutyric acid and provide pre-liminary chemical evidence for the presence of PHB in chemos tat cultures of Legionella pneumophila. The PHB was located in electron-dense intracellular inclusions, which fluoresced bright yellow when stained with the lipophilic dye Nile red. A Nile red spectrofluorometric assay provided a more accurate and reliable determina-tion of the PHB content. L.pneumophila accumulates significant intracellular reserves ofPHB, which promote its long-term survival under conditions of starvation u.

PHB has been detected in the cytoplasmic membrane of Escherichia coli. E.coli cells incorporate PHB into their plasma membranes under growth-limiting condi-tions and during competence development. Recently, 3 genes responsible for the synthesis of PHA have been cloned from Alcaligens eutrophus and expressed in

E.coli 11 • PHB was recovered by using a sodium hypochlo-rite solution or by using dispersions of a sodium hy-pochlorite solution and chloroform from A.eutrophus and a recombinant E.coli strain harboring the A.eutrophus PHA biosynthesis genes. A recombinant strain of Es-cherichia coli was used to produce poly (4-hydroxybu-tyric acid), P( 4HB), homopolyester by fed-batch culture in M9 mineral salts medium containing glucose and 4-hydroxybutyric acid as carbon sources. The discovery of poly (3-hydroxybutyric acid), poly (3HB), as astor-age compound in Ba~illus megaterium was made in 1926, D(-)-3-hydroxybutyric acid (3HB) remained the only known constituent of bacterial poly-hydroxy-alkanoic acids (PHA) 14•15 until constituents other than 3HB were reported in the sixties and seventies. However, these re-ports were either based on preliminary chemical analy-sis or they failed to attract the deserved attention . In the early eighties, 3-hydroxyvaleric acid (3HV) and 3-hydroxyhexanoic acid (3HHx) were incidently detected as constituents of PHA accumulated by axenic cultures of Bacillus sps. or Alcaligenes eutrophus and 3-hydroxyoctanoic acid (3HO) was detected as a constitu-ent of PHA accumulated by Pseudomonas oleovorans 16•

Molecular and Biochemical Properties of PHA and PHB

Poly-hydroxy alkanoates (PHAs) are polyesters of various hydroxy-alkanoates. More than 100 different monomer units like 3-0H acyl monomers, have been identified. PHB has lowest molecular weight and is most common in nature. Their molecular weight can be up to 2 million, i.e. , 20,000 monomers/polymer molecule. The monomer units of PHA are all in D-(-) configuration owing to the stereospecificity of the biosynthetic en-zymes.

There are three enzymes present in A.eutrophus for PHA biosynthesis. These are - PHA synthase, ~ketothiolase and reductase. Natural producers also have PHA depolymerase that degrades the polymer and uses the breakdown metabolites for cell growth 17 •

Metabolism of PHB occurs as: PHB ---7 D (-)hydroxybutyric ac id ---7 acetoacetate or acetoacetyl CoA.

Properties of PHB

(i) PHB is water insoluble and relatively resistant to hydrolytic degradation. This differentiates PHB from most other currently avai lable biodegradable

438 J SCI IND RES VOL 59 JUNE 2000

plastics, which are either water soluble or mois-ture sensitive.

(ii) PHB shows good oxygen permeability.

(iii) PHB has good UV resistance but has poor resis-tance to acids and bases.

(iv) PHB is soluble in chloroform and other chlorinated hydrocarbons.

(v) PHB is bio-compatible and hence is suitable for medical applications.

(vi) PHB has melting point 175°C and glass transition temperature 15°C.

(vii) PHB has tensile strength of 40 Mpa, which is close to that of polypropylene.

(viii) PHB sinks in water, whereas polypropylene floats . But sinking ofPHB facilities its anaerobic biodeg-radation in sediments.

(ix) PHB is nontoxic .

As many as 91 different HA have been detected as constituents of biosynthetic PHA. That all these HA co-enzyme A thioesters are substrates of PHA synthases was concluded in most cases from the occurrence of the corresponding HA in the polyesters and from homolo-gies to known pathways rather than by enzymatic stud-ies. Many more substrates of PHA synthases and there-fore new constituents ofbiosynthetic PPHA could prob-ably be detected by further in vitro studies. Recently, lactyl-CoA was shown to be a poor substrate for the PHA synthases of some bacteria, indicating the incorporation of lactic acid into PHN8•

Biosynthesis of PHB The biosynthesis of PHB in most bacteria is initiated

by the condensation of two molecules of acetyl-CoA by 3-ketothiolase to form aceto-acetyi-CoA, which is re-duced in an enantiomerically selective reaction by aceto-acetyi-CoA reductase to R-(-)-3 hydroxy butyryl-CoA and is incorporated into PHB by PHB synthase. The ac-etoacetyi-CoA reductase involved in PHB synthesis is generally considered to be specific for NADPH, although in some bacteria the enzyme has been reported to have activity with NADH as well as NADPH. CCII92 cells retain the capacity for PHB synthesis when they differ-entiate from free-living cells into the bacteroid form' 9.

Biosynthesis of PHA Three metabolic phases of the biosynthesis of PHA

in bacteria can be distinguished (Figure 2).

PHASE I PHASE II (Membrane-associated (Soluble proteins)

· protein)

PHASE Ill (Granule usoclated

protein)

Carbon Sourc

~ Carbon Hydroxy ac:yi..Co souz /

I Catabolism I

Figure 2-Three relevant phases of biosynthesis of PHA bacteria16

Phase I -A carbon source suitable for biosynthesis of PHA must enter the cell from the environment.

Phase II- Anabolic or catabolic reactions - or both- convert the compound into a hydroxyacyl coenzyme A thioester which is a substrate of the PHA synthase.

Phase III- PHA synthase, which is the key enzyme of PHA biosynthesis of these thioesters as substrates and catalyzes the formation of the ester bond with the concomitant release of coenzyme N 6 .

The PHA synthase from T.pfennigii exhibited the most unusual substrate range among the PHA synthases in-vestigated so far. However, this only became obvious if this PHA synthases was heterologously expressed in a different physiological background20.

Most PHA consist of two or more HA. As outlined recently21 the metabolic reactions occurring in Phase II may result in the formation of various different HA co-enzyme A thioesters that are used as substrates by the rather unspecific PHA synthases, thus giving rise to the synthesis of co-polyesters.

The potential for the production of new PHA seems to be limited by the availability and costs of chemicals which can be provided as precursor substrates to the bacteria, rather than by the substrate range of PHA synthases. Therefore the chances of obtaining PHA with new HA or with an unusual combination of HA in the future, will depend on the successful screening for bac-teria which synthesize these precursor substrates endog-enously from simple and cheap carbon sources. Recently, bacteria were isolated which synthesize poly(3HB-co-3HV) or PHA from unrelated substrates2 1•

MCL

Despite the excitement of more than 90 different con-stituents of biosynthetic PHA, it may be pointed out that the commercial exploitation of this variety remains lim-ited. At present, only very few PHA are available in suf-ficient amounts to allow the evaluation of the physical,

KALlA et al.: BIOPLASTICS 439

PHASE I

H, + CO, Acetate Butyrate Poly hydroxy 1 ,----', \ /' butyrate !

p;j~~i!iii-.-.~illiffmoioplastics) i

I I / /',\ ~~--, J? ~ I 1 / " / , \ 1 \ , t / ,~ Ill Acetogenesis I ' \ 1 1 A' Homoacetogenic & ! '\~;~)~t[O~ Hydrogenogenlc bacteria

I PHAS;l~~G[E~r::,.,.~ tt..:-::'ip=::-:-:-:-=-7=:-:::=-::,-----,

! I I

I

I ! i

I ~--·------------ -·-- - ---·./

----;:. Primary Fermentative Process -M·-- Tertiary Fermentative Process {~ Secondary Fermentative Process

Figure 3 -Anaerobic biodegradation of waste biomass

chemical, and biological material properties of these polyesters16.

Production of Bio-plastics by Anaerobic Digestion of Biological Wastes Anaerobic Digestion

Anaerobic digestion process is a multiple stage pro-cess (Figure 3). Complex biomolecules are hydrolysed into relatively simpler compounds. A second group of acedogenic and acetogenic bacteria metabolise the hy-drolytic products in lower volatile fatty acids22-24, hy-drogen and carbon dioxide. In the ultimate stage, methanogens convert these intermediates into methane25 •

Various process parameters for anaerobic digestion numerous bio-wastes of diverse origins have been es-tablished26: Municipal market wastes27·2K viz. leaves and stalks of radish, cauliflower, cabbage, knol khol and ba-nana29, Food Corporation of India waste viz. , damaged food grains30, National Dairy Development Board Fruit

Table 3 - PHB accumulation in microorganisms x

Organisms with PHB , i.e., (PHA) polyhydroxy alkonates accumulation

Alcaligenes eutrophus Azospirillum Azotobacter Baggiatoa Leptothrix Methylocystis Pseudomonas Rhizobium Rhodobacter

Per cent of dry wt of cell

96 75 73 57 67 70 67 57 80

and Vegetable Unit Waste viz. pea-shells, HPMC. Fruit Processing Plant waste viz. Apple Pomace, and Palm Oil Mill Effluent31·32.

Poly hydroxy butyrate (PHB) is the key ingredient for bio-plastic production. The precursor to the formation are lower volatile fatty acids33. Poly ~-hydroxy butyrate (PHB) accumulates as energy reserve material in many microorganisms like Alcaligenes, Azotobacter; Bacillus, Nocardia, Pseudomonas, and Rhizobium.

PHB has physical properties comparable with polypro-pylene (PP). PHB comprises repeat units of CH(CH3)-CH2-CO-O. The difference is that PP shows insignifi-cant degradation while PHB shows complete degrada-tion . PHB sinks while PP floats . Therefore, degradation is easy as sediment. Alcaligenes eutrophus and Azoto-bacter beijerinckii can accumulate up to 70 per cent of their dry weight of PHB as given in Table 3. These mi-croorganisms can produce the polymer in environment of N and P limitation. At least 40 to 50 per cent of the dry weight of this polymer is required for making the process commercially viable. Extraction ofPHB is done by using solvents like halogenated hydrocarbons and purification is done. Molding and extrusion of dried cells is directly possible when PHB contents are high. Lot of work has been done on engineering polymeric proper-ties of PHB. However, PHB is suitable for specialized areas like biomedical use, specially coatings8•

Biowastes From activated sludge from a domestic sewage plant,

Wallen and Rohwedder isolated PHA consisting of 3HB, 3HV, and 3HHx, there was also some evidence for the presence of traces of3-hydroxyheptanoic acid34. Analy-sis of activated sludge from a sewage treatment plant in Veberod (Sweden) revealed PHA, consisting of 3HB, 3HHx, and 3HO.

440 J SCI JND RES VOL 59 JUNE 2000

PHA cons1sttng of 3HB, 3HV, 3-hydroxy-2methylbutyric acid, and 3-hydroxy-2-methyl valerie acid were obtained from activated sludge from a domes-tic sewage plant in Tokyo, which was withdrawn from the plant and subsequently cultivated in complex me-dium in a bio-reactor. PHA isolated from an estuarine sediment contained, in addition to 3HB, which contrib-uted to only 30 per cent (w/w) of the constituents, at least five other 3HA.

Biodegradable thermoplastics, poly hydroxy alkanoates (PHAs) were produced from municipal sludge35 and Palm oil Mill effluent'637. Biodegradable components in municipal sludge and other organic wastes are digested under anaerobic conditions by ac idogenic bacteria into volatile fatty acids such as acetic, propi-onic, butyric acids, and other soluble organic com-pounds3x39. It has been reported that Alcaligenes eutropus can utilize individual volatile fatty acid and polymerize the acid into PHAs as its carbon and energy reserve un-der nitrogen or oxygen limited conditions39.42.37. Many other bacterial strains are also reported to produce PH As under adverse conditions with different PHA yieJd36.43·44 • Palm oil mill effluent has been employed as feed mate-rial for anaerobic digestion, the intermediate acetic acid was then used as a substrate in the fed-batch production of polyhydroxyalkanoate (PHA) by Alcaligenes eutrophus and by Rhodobacter sphaeroides strain IFO 12203.

PHAs have been extracted with hot chloroform from activated municipal sludge and detected in marine sedi-ments34. Biodegradable "enviroplastic" is developed by Planet Technologies, USA. Combinations of various car-bon and nitrogen substrates were used to study PHB ac-cumulation and H2 evolution by Rhodobacter sphaeroides45 • PHA was continuously produced by Rhodobacter sphaeroides when sludge particles from anaerobically treated POME were removed36·37·40 .

Biodegradability Tests Generally, ASTM recommended microbial cultures

are used. Systematic studies, employing wide range of microbes from diverse ecological habitats including hydrocarbon degraders has not been reported. Standard methods for comparison have not so far been estab-lished47.

Biodegradability test is dependent upon the objec-tives, e.g., (i) To determine the resistance of polymers from all

sources, a mixed inoculum is preferred.

(ii) If the environment where the polymer is expected to be exposed,_ an inoculum derived from it is an ap-propriate choice.

(iii) If the end is not known, inocula preferred from soil, humus, domestic sewage or a mixture of known or-ganisms proves effective.

Generally the biodegradability test is carried out by soil burial of the specimen and monitoring various physi-cal, mechanical, and chemical properties of the test speci-men as a function of time. Following tests have been in use47:

(i) Warburg Test (specification 301) - In this test the quantity of oxygen consumed during biochemical degradation (Biochemical Oxygen Demand, BOD) of a sample is measured as a function of time to the theoreti-cal quantity of oxygen required for complete oxidation of the specimen kept under controlled test conditions. The test samples may be buried under soil or kept out-doors .

(ii) ASTM D-5338 - The test samples are buried under soil of controlled humidity and composting con-ditions and loss of physical form, weight, mechanical strength is measured as a function of time. Biodegrada-tion of samples may also be monitored by other test methods viz. a) change of molecular weight due to deg-radation by gel permeation chromatography, b) viscom-etry, c) change of carbonyl group content due to degra-dation by IR Spectrophotometry, d) C0

2 evolution by

Liquid Scintillation Counting and e) Colony growth, etc.

(iii) Microbial Testing - Pure cultures or enzymes and pure substrates can not be compared to water, sew-age and oil environment so as a direct application is highly questionable3. Degradation pathways occurring in one ecosystem may not be identical with those found in an other.

(iv) Biodegradation on Solid Medium (Agar plate methods) 48·49 - Most common test for assessing the bio-degradability of polymers . It consists of: (l) Selection of suitable specimens, (2) Inoculation with microbes, (3) Incubation, (4) Testing for changes in the specimen. Agar plates as per ASTM standard. High melting agar em-ployed for thennophiles. Plastic sample (2x2cm) was placed on agar plate and inoculated with microbial sus-pension. Glucose and NH4 N03 were used as C and N

KALlA et at.: BIOPLASTICS 441

Table 4- Fungi recommended InternationalJy for biodegradation tests50·"

Russian proposal AFNOR NF X 41-514 USSR-I 1958 Aout 1961

Chaetomium Chaetomium globosum globosum

MemomielJa echinata

Trichoderma Tl

Stachybotyrys Stachybotyrys atra atra

Pencillium Pencillium bravicompactum bravicompactum

Pencillium Pencillium cyclopium cyclopium

PencilJium funiculosum

Paecilomyces Paecilomyces varioti varioti

Aspergillus Aspergillus amstlodami amstlodami

AspergilJus niger

Aspergillus flavus Myrothecium rerrucarcia

source. Microbial growth observed for two weeks (Growth indicates non-toxicity of plastic). In controls, C and N were omitted to check if plastic can replace these sources .

(v) Biodegradation on Broth- Physical changes like pH, weight loss (of the sample) and biomass generation. were recorded. Fungi is recommended in some of the most important internationally quoted biodegradation tests are given in Table 4. These microbes were initially employed for testing the resistance of cotton textiles. Fungi with an established ability to attack hydrocarbons are the right candidates, along with cellulase utilizers .

Since the number of biological parameters outnum-ber the individual interspecies differences among most

ISO R 846 ASTM D 1924 1968 197

Chaetomium Chaetomium globosum globosum

PulJularia pullulans

Trichoderma TI Trichoderma spp.

Pencillium Pencillium funiculosum funiculosum

Paecilomyces varioti

Aspergillus Aspergillus niger niger

of the soil organisms, adequate proof of a cause and ef-fect relationship can only be provided with the pure cul-tures , as individual or a consortia.

(vi) Radiocarbon 14C Studies- The use of 14C con-stitutes one means to distinguish C0

2 produced from

the polymer and other sources52·53 14C polyethylenes 2 per cent/y HDPE film reduced C0

2 evolution on re-

moval of low mol wt components . .

(vii) Soil Burial Method- It lacks reproducibility due to climatic factors and lack of control over micro-bial population Clendining et al.54 enriched microbes from soil buried polymer. Ennis and Kramer55 outlined a rapid method of (respirometric) to measure oxygen ab-sorption by microbes and weight loss method56.

442 J SCI IND RES VOL 59 JUNE 2000

Table 5 -Industrial production of PHA's and other biodegradable plasticsx

Company

Berlin Packaging Corp. (USA) Bioscience Ltd. (Finland) Bio ventures Alberta Inc. (Canada) Metabolix Inc. (USA) Monsanto (USA) Polyferm INC. (Canada) Zeneca Bio-products (UK) (former ICI,UK) Zeneca seeds (UK) Petrochemia Danubia (PCD)

Areas of interest

Marketing Medical applications Production in recombinant E. Coli. Production in transgenic plants Production from cheap substrates.

Production by A. eutrophus

Production in transgenic plants Production using A. [actus.

Table 6 - Manufactures of biodegradable plasticsH

Manufacturer

Warner- Lambert Company, USA

2 Amke plastics, USA 3 Air products and chemicals , USA 4 Agro Chemical Company, USA 5 Novamont, Italy

6 Cabot Europe Ltd, France 7 Exxon Chemicals, Belgium 8 Phone-Poulenc Chemie, Belgium 9 Hoechst, Germany I 0 Nova Corporation, Canada JJ . American Cyanide Co. Ltd, USA 12 ICI, USA 13 Ethicon Inc,

(viii) Visual Rating- It is not a very desired method and is largely a measurement of the effect of the plastic upon the micro-organisms and vice versa. Also, a non-filamentous organism may cause much more severe deg-radation than a filamentous organism like fungus.

(ix) Ammonia Production- Bacteria release ammo-nia by means of endogenous respiration of their proteins in the absence of an utilizable carbon source57• Values of ammonia produced by PVC samples show an excel-lent inverse correlation to weight loss of the file, and become the basis of a rapid method for assessing biode-gradability.

(x) Non-biological Methods- These include changes in mechanical, physical and chemical properties like flex-

Product (Trade name)

Noven (Molding grade) Polyethylene

Mater-B I (Molding grade) Cornplast (Master batch)

Poly glycoside poly lactide and their co-polymers

ibility, elasticity or tensile strength, weight loss has been found to correlate most closely5R. Loss of transparency and loss of good di-electric properties of films are the basis of a rapid method for assessing biodegradability.

International Status - US chemical company Monsanto is already commercially producing (Table 5 and 6) a plastic material called "biopol" by fermentation of the bacterium Alcaligenes eutrophus. But the cost of production is highR.

Markets for Biodegradable Polymers Man-made polymers have been continuously and sys-

tematically improved to make them more stable, me-chanically stronger and resistant to chemical and envi-ronmental conditions. Rot-resistance and rust-proof char-

KALlA et al.: BIOPLASTICS 443

Table 7- Biodegradable polymer productsM

Field

Agriculture

2 Horticulture

3 Packaging

4 Domestic

5 Hospital

Product

(i) Fibre (ii) Film

(i)Film (ii) Moulded item

(i) Film

(ii) Blister packaging and bubble sheets

(i) Films

(ii) Moulded products

(i) Moulded products

acteristics of polymers have increased their demand and popularity. Other features that have made the usage of synthetic polymers in our daily life at an accelerated rate have been their low price, ready availability, wide range of colouration, and ease of fabrication in all the desired shapes. However the disposal of the used product is be-coming increasingly difficult. Legislation have been, however, enacted in many developed countries where use of biodegradable plastics in certain applications is mandatory. It is thus estimated that by the end of this century biodegradable plastics will account for about 3 per cent of all plastic wastex. Some of the biodegradable polymers are available in market (Table7) .

Present Status of Commercial Biodegradable Plastics

Although, plastics are in use and demand, however the market for degradable plastics is relatively very small.

Application

Netting Mulching controlled release of agro-chemicals and micronutrients, fertilizers.

Nursery bags, Plant cover and plant holders .

Packaging of perishable foods, dairy products, fruits,

Vegetables, hosiery, etc. Fragile goods packaging

Shopping bags, composting bags, Diapers, feminine products, hygiene, garbage bags

Food containers, vegetable crates, food service items

egg-boxes, toys, pens, cutlery and cups, razors, containers for beverages, etc.

Disposable needles and syringes, sutures,

surgical gloves, .gowns, etc.

It represents less than 20 per cent of the total demand for plastics in the developed countries like the US or Japan. In India the demand may be less than 5 per cent. The major reason for this is the recycling of plastics. In India, almost every plastic item- films or molded prod-ucts, domestic or municipal garbage are hand-picked, washed, dried, ground and recycled . Disposal of plas-tics will pose a real problem only if collection and pro- . cessing cost of the reclaimed plastics is much more than that of the virgin material. Consequently, markets for degradable plastics will then be bright.

Starch-based plastic products will enjoy the attention of plastic processing industry for biodegradation and cost reduction, since the price of starch is almost lf5t11 the cost of poly ethylene. Even partial replacement of the latter by the former by a simple technology appears to be commercially viable . Indian Institute Technology (liT), Kharagpur has shown that incorporation of starch

444 J SCIIND RES VOL 59 JUNE 2000

into LDPE up to 10 per cent only marginally affects the strength properties of the plastics. The hydrophobicity of the starch-based plastic products could be controlled by addition of suitable additives.

It is being realized that the use of long-lasting poly-mers for short-lived applications is not entirely justified, especially when increased concern exists about the pres-ervation of living systems. The elimination of waste plas-tics is therefore of intere~t in surgery, hygiene, catering, packaging, agriculture, fishing, and environmental pro-tection application 1•

Acceptance of biodegradable polymers is likely to de-pend on four unknown:

(i) Customer response to costs that today are generally 2-4 times higher than for conventional polymers,

(ii) possible legislation (particularly concerning water-soluble polymers),

(iii) The achievement of total biodegradability, and (iv) The development of an infrastructure to collect,

accept, and process biodegradable polymers as a generally available option for waste disposaP9.

Thus the bio-plastics of the future will be produced from renewable sources, will have a low energy content, .and will display in-use properties similar to those of con-ventional plastics.

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