Production of biodegradable thermoplastics from municipal sludge by a two-stage bioprocess

ELSEVIER Resources, Conservation and Recycling 19 (1997) 151- 164 drecgcllng

Production of biodegradable thermoplastics from municipal sludge by a two-stage bioprocess

Samuel Lee, Jian Yu*

Department of Chemical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

Received 30 May 1996; revised 25 July 1996; accepted 1 August 1996

Abstract

Biodegradable thermoplastics, polyhydroxyalkanoates (PHAs), were produced from municipal sludge in a two-stage bioprocess; anaerobic digestion of sludge by thermophilic bacteria in the first stage and production of PHAs from soluble organic compounds in the supernatant of digested sludge by Alcaligenes eutrophus in the second stage. Local municipal sludge of 0.3-S wt % dry solids containing 50-70% volatile solids was digested under thermophilic (50-65°C) and anaerobic conditions producing a mixture of fatty acids and other minor fermentation products. The residual sludge contains about 50% of the original volatile solids. Volatile solids reduction and formation of soluble organic compounds from sludge were investigated under different temperature, pH, hydraulic (solid) retention times. More soluble organic compounds were produced at high temperature (65°C) and pH ( > 5.5) with little change in volatile solids reduction. In the second stage, harvested from a nutrient-rich culture, A. eutrophus produced PHAs from the soluble organic compounds in the supernatant of digested sludge under aerobic and nitrogen-limited conditions. The amount of PHAs accumulated in A. eutrophus accounted for 34% of cell mass, comparable to the amount of PHAs produced from pure volatile fatty acids (33% of cell mass). About 78% of the total organic carbon in the supernatant was consumed by A. eutrophus. The conversions of four major acids in the supernatant by A. eutrophus were found to be 87.6% (acetic acid), 62.6% (propionic acid), 56.8% (butyric acids) and 32.0% (valeric acids), respectively. The sludge PHA is a co-polymer containing mainly C, monomers (74 wt %) and has a melting point of 167°C which is 9°C lower than that of poly-3-hydroxybutyrate (PHB). The sludge thermoplastic and PHB had a similar biodegradability in soil, 22-27% in 5 weeks. In sludge suspensions, 70% of sludge plastic was decomposed and utilized by anaerobic and aerobic bacteria in 6 weeks. Copyright 0 1997 Elsevier Science B.V.

* Corresponding author. Tel.: + 852 23587135; fax: + 852 23580054; email:[email protected].

0921-3449/97/$17.00 Copyright 0 1997 Elsevier Science B.V. All rights reserved PII SO921-3449(96)01157-3

152 S. Lee, J. Yu /Resources, Conservation and Recycling 19 (1997) 151-164

Keywords: Anaerobic sludge digestion; Biodegradable plastics; Polyhydroxyalkanoates; Mu- nicipal sludge

1. Introduction

Disposal of municipal wastes including sewage sludge and plastic materials poses challenges to municipalities worldwide. Sludge disposal may cost US$l50-450/tori dry solids depending on the methods traditionally used such as landfilling after dehydration, incineration and land application [l]. One hundred fifty four tons of sewage sludge per day are disposed of at landfill sites in Hong Kong and more sludge will be produced under stricter wastewater control regulations [2]. With the phase-out of ocean dumping and lack of landfill sites, coastal communities like Hong Kong have to find innovative ways due to the limited options available. Anaerobic digestion is a widely used sludge treatment process which stabilizes the sludge (lessening odor problem), reduces the solids content for landfilling, and destroys pathogen under thermophilic conditions [3]. Sludge treated at 65°C for up to 3 h can be safely applied as Class A biosolids to a lawn or home garden [4] It has been understood that anaerobic digestion of sludge goes through acidogenesis (acids-producing) and methanogenesis (methane-producing). Acidogenesis of sludge produces volatile fatty acids and other soluble organic compounds which are further utilized by methane-producing bacteria [5].

Over the past decades, the intrinsic resistance of plastic materials to degradation has been increasingly regarded as a source of environmental and waste management problems. Plastic materials account for about 20% by volume of municipal solid wastes and reduce the capacity of precious landfill sites [2,6]. The development and production of degradable plastics is rapidly expanding due to, in part, the growing legislation in developed countries aimed at baring the use of non-degradable plastics in a variety of consumer products [7]. There is growing interest in the group of polyhydroxyalkanoates (PHAs), the polyesters made entirely from bacterial fermentation [8]. These biodegradable thermoplastics can be processed like synthetic ones such as polyethylene and polypropylene. One such commercial PHA is a random copolymer, p(HB-co-HV), of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), produced by bacterium Alcaligenes eutrophus from glucose and propionic acid [9]. The major drawback of PHB and other bacterial PHAs is their high production cost including the raw materials, making them substantially more expensive than synthetic plastics. Cost of biodegradable plastics is restricting their large-scale use in consumer products [9].

Biodegradable components in municipal sludge and other organic wastes are digested under anaerobic conditions by acidogenic bacteria into volatile fatty acids such as acetic, propionic, butyric acids and other soluble organic compounds [lO,l 11. It has been reported that Alcaligenes eutrophus can utilize individual volatile fatty acid and polymerize the acid into PHAs as its carbon and energy reserve under nitrogen- or oxygen-limited conditions [12,13]. Many other bacterial strains

S. Lee, J. Yu / Resources, Conservation and Recycling 19 (1997) 151-164 153

are also reported being able to produce PHAs under adverse conditions with different PHA yields [S]. PHAs have been extracted with hot chloroform from activated municipal sludge and detected in marine sediments [14,15]. Converting biodegradable components in municipal sludge under thermophilic conditions to volatile acids and further into PHAs has its merits for sustainable development and waste management such as less and safer sludge to be handled, less methane produced in landfill sites, lower cost for sludge disposal if it can be partially utilized as raw materials to produce valuable products, and production of true biodegradable thermoplastics. This paper reports our investigation on a two-stage process through which biodegradable components in municipal sludge were converted to PHAs.

2. Materials and methods

Sludge was taken from aeration tank and sedimentation tank in a local municipal wastewater treatment center. On average, the sludge contained 60% of volatile solids measured as described below. Acetic, propionic, butyric, valeric and crotonic acids were purchased from Sigma (St. Louis, MO, USA). Poly-3-hydroxybutyrate (PHB), poly-3-hydroxybutyrate-co-3-hydroxyvalerate (8% PHV) and other chemi- cals were purchased from Aldrich (Milwaukee, WI, USA) except those indicated.

2.1. Anaerobic sludge digestion

Two-liter sludge (5 g dry solids/L) was adapted from 25 to 50°C over 30 days under anaerobic conditions, and then transferred to a 3 L anaerobic digester as shown in Fig. 1. The digester was completely mixed and equipped with pH control. Fresh sludge suspension (0.3-5 wt % total solids), stored in a cooler, was continu- ously pumped into the digester at a predetermined hydraulic retention time (HRT).

LiId_Ail”l l!!rn r Sludge Waterbath Feed Anaerobic Clarifier Recycle

Effluent Cooler Digestor

Fig. 1. Schematic diagram of the bench-top facility for anaerobic sludge digestion.

154 S. Lee, J. Yu / Resources, Conservation and Recycling 19 (1997) 151-164

The effluent from the digester first went to a clarifier and then overflowed into the effluent tank. The sedimentary sludge solids in the clarifier was transferred (every 12 h) to a sludge recycle tank and pumped back to the digester. The solid retention time (SRT) was calculated from the total solid content in the digester and the solid discharge rate. Therefore, the controllable parameters included pH, temperature, HRT (SRT) and inlet solid content. Samples were taken from the digester, effluent tank and recycle tank for measurement of soluble products including fatty acids and total organic carbon (TOC), and of residual solids including volatile solids and total solids. Reductions in volatile and total solids of sludge were determined from the solid contents in the inlet and outlet streams. The investigation used mainly a relatively low solid-content sludge (0.3-0.5 wt %) for reliable operation of the bench-top facility. The effluent liquid from the digester was centrifuged at 5000 g for 10 min, and the supernatant was used for PHA production by A. eutrophus.

2.2. PHA production by A. eutrophus

A. eutrophus (ATCC 17699) was maintained on a agar slant containing per liter; fructose 10 g, yeast extract 10 g, peptone 10 g, meat extract 10 g, agar 15 g and was cultivated in a 2 L fermenter (Gallenkamp, UK) for cell mass production with little PHA accumulation in the cells. In this stage, a nutrient-rich culture medium was used containing per liter; 10 g peptone, 10 g yeast extract, 5 g meat extract and 5 g (NH&SO,. The cultivation was controlled at 30°C 50 rpm agitation, 300 cm3 air (STP)/min and pH 7.0. The cells, after 24-h cultivation, were harvested with centrifugation at 10000 g for 10 min at 4°C. After being washed with 0.8% NaCl solution to remove residual nitrogen and other nutrients, the cells were cultivated again in the supernatant of digested sludge in the 2 L fermenter under the conditions as above but without pH control. For comparison, mixtures of pure propionic and butyric acids were also used for PHA production by the harvested A. eutrophus cells. Because long chain acids have higher yield of PHAs than acetic acid does [12], acetic acid was not added into the mixtures though it was one of the major products from sludge digestion. Minerals were added to avoid cell lysis in pure acid cultures (per liter); 4.8 g Na,HP0,.2H,O, 2.65 g KH,PO,, 0.4 g MgSO,. 7H,O and 1.0 mL trance elements [l I]. After 48-h cultivation, the cells were harvested with centrifugation, and freeze dried for polymer extraction with hot chloroform in a Soxhlet apparatus. PHAs in the chloroform solution were further purified by precipitation with n-Hexane.

2.3. Biodegradation of PHAs

Plastic films were prepared by dissolving commercial and sludge PHAs in hot chloroform and pouring the solution into glass plates for solvent evaporation. Plastic films (0.1-0.3 mm thick) formed on the bottom of the plates were used to test the biodegradability of PHAs in sludge suspension, sea water and soil. The weight loss of plastic films was monitored.


Table 1 Solids and soluble TOC in anaerobic sludge digestion

Inlet Digester

Total solids, g/L 4.5 6.4 Ash, g/L (%) 2.3(51) 2.9(46) Volatile solids, g/L (%) 2.2(48) 3.5(54) Soluble TOC, g/L 0.01 0.17

Process conditions, SRT 1 day, HRT 0.5 days, 65°C (pH 4).

Outlet

3.1 2.2(71) 0.9(29) 0.17

I/O change

- 32% 0%

-55% 0.16

2.4. Analysis

Total solid content of sludge was determined by dehydration of sludge to constant weight at 105°C over night, and volatile solid content was measured after combustion of the solid at 550°C. Total organic carbon (TOC) in the supernatant of digested sludge was measured using a total organic carbon analyzer (Shizmado TOC-5000, Japan). Volatile organic compounds were assayed with a gas chro- matography (Hewlett Packard 6890, Wilmington, USA) equipped with a NUKOL fused silica capillary column (Supelco, Bellefonte, USA) and flame ionization detector (FID). The volatile organic compounds were eluted with a temperature program of 2°C increase per minute from 130 to 150°C and helium 2 mL/min. The compositions of PHAs were assayed by hydrolyzing the polyesters in 1 mL of 98% sulfuric acid for 1 min at 90°C and analyzing the diluted samples with the GC as described above. Melting properties of the thermoplastics were detected with a differential scanning calorimeter (DuPont 29 10, Newtown, USA).

3. Results and discussion

3.1. Anaerobic sludge digestion

The major concern with sludge digestion was volatile solids reduction and production of soluble organic compounds from the digested solids. At steady state of a typical run as shown in Table 1, the amounts of ash fed into and leaving the continuous flow digester were the same. About 55% of volatile solids was digested, and hence total solids reduction was 32%. The digested volatile solid was partially converted to soluble organic compounds (soluble TOC increased from 0.01 to 0.17 g/L), partially to gaseous products, and partially utilized for maintenance and growth of thermophilic anaerobes. Depending on hydraulic retention time (HRT) controlled, the volatile solids content of the active biomass in the digester was 5-25% higher than that of the feeding sludge due to the growth of thermophilic anaerobes. The amount of volatile solids converted into gaseous products (CH,, CO,) was not measured in this study. The ratio of volatile solids reduced to the amount of soluble TOC formed was 8, a value much higher than 2.5 of an empirical


biomass formula (CH,O),. This indicates that considerable amount of digested organic solids was not released into the liquid as soluble TOC under the conditions. The soluble TOC from sludge digestion included acetic, propionic, butyric, valeric acids and other soluble organic compounds (Fig. 2). With a higher inlet solid content (51 g/L or 5.1 wt %), the percentage of volatile solid reduction did not change very much (52.5%), but the soluble TOC; content went up to 2.4 g/L.

The effects of temperature, pH, and HRT (SRT) on volatile solids reduction (VSR) and soluble total organic carbon (TOC) were investigated with low-solid content sludge (0.4 wt % total solids). In general, volatile solids reduction was quite consistent and not affected by these factors considerably, ranging from 40 to 55%, as shown in Fig. 3-5. This consistency might be attributed to the consistency of percentage of biodegradable component in the sludge. Therefore, 50% volatile solids reduction is achievable and 30% total solids can be reduced if typical sludge contains 60% volatile solids. The soluble total organic carbon, however, was influenced very much by the process conditions. After the temperature was increased from 50 to 65°C the soluble organic compounds rose from 50 to 200 mg TOC/L as shown in Fig. 3. A greater effect was observed when pH was changed from 4.1 to 5.4 as demonstrated in Fig. 4. The soluble TOC was increased from 80 to 480 mg/L correspondingly. At this pH level (5.4), the ratio of volatile solids reduced to the amount of TOC formed had a value of 3.2, compared with 8 at pH 4 (Table 1). This may indicate a more favorable pathway of digested biomass to

I /

6 8 miq

Fig. 2. GC chromatogram of the supernatant of digested sludge; acetic acid (AH), propionic acid (PH), iso-butyric acid (i-BH), butyric acid (BH), iso-valeric acid (i-VH) and valeric acid (VH).

S. Lee, J. Yu /Resources, Conservation and Recycling 19 (1997) 151-164 157

300 100

-- 80

y 200 -- h -- 60 ? -E 5

150 --

8 -- 40 % > + 100 --

-- 50 20 --

0 I I I I I I I I 0 45 50 55 60 65 70

Temperature (C)

Fig. 3. The effect of temperature on volatile solids reduction (VSR) and soluble total organic carbon (TOC) in anaerobic sludge digestion; pH 4 and HRT 2 days (SRT 1.5-2 days).

soluble organic compounds at pH 5.4 than at pH 4. A longer hydraulic retention time led to a lower TOC concentration in the supernatant with little effect on VSR as shown in Fig. 5, a fact that soluble organic compounds were further converted into methane by methanogenic bacteria. Therefore, HRT is a parameter that can be controlled for the formation of acidogenic products. The significance of the parameters to soluble TOC formation is; pH > temperature > HRT (SRT).

3.2. PHA formation

Negligible PHAs was found in harvested A. eutrophus cells from the first nutrient-rich cultivation; the amount of PHAs extracted by hot chloroform ranged from l-3% of cell mass. Cultivation of the cells in the supernatant of digested sludge under aerobic and nitrogen-limited conditions raised the PHA content in the cells to 34% as shown in Table 2. Correspondingly, the soluble organic compounds in the supernatant dropped from 0.62 to 0.13 g TOC/L, a conversion of 78%. Furthermore, by comparing the contents of fatty acids in the supernatant (Fig. 2) before and after PHA production, the conversions of four major acids by A. eutrophus were found to be 87.6% (acetic acid), 62.6% (propionic acid), 56.8% (butyric acids) and 32.0% (valeric acids), respectively. It seems that in a mixture of fatty acids the longer the chain of an acid, the slower its take-up by the cells, The

158 S. Lee, J. Yu / Resources, Conservation and Recycling 19 (1997) 1.51-164

final cell density in Table 2 was determined by the culture volume and the total freeze-dried cell mass harvested. The amount of PHA was the precipitate mass recovered from chloroform solution, which actually included all possible losses during the extraction and purification. A. eutrophus could produce PHAs from the supernatant of digested sludge as well as from the mixture of pure propionic and butyric acids. The similar PHA content in cells and TOC conversion in solution indicate that the bugs could utilize these two types of carbon source with the same efficiency. A further improvement on polymer production was possible with a two-step acid feeding control as demonstrated in Table 2.

3.3. Biodegradation of PHAs

Fig. 6 shows that plastic films made from sludge PHAs were degraded quickly (about 70% mass in 6 weeks) by aerobic and anaerobic bacteria in sludge suspensions, but relatively slowly in soil and sea water (lo-20% mass in 6 weeks). This fact implies that the degradation of PHAs depended on microbial activity since the microbes in soil were less than those in sludge suspensions, and also had poorer contact with the polymers. It was reported recently that two extracellular bacterial enzymes responsible for PHA degradation had been identified [16]. A control test in tap water showed no degradation at all. Degradation of plastic films made from commercial polyhydroxybutyrate (PHB) and sludge PHAs (SPHAs) in soil was also

6oo I loo 500 -- *

i+ 400 -- ?n g 300 --

8 I- 200 --

100 -- -e TOC +VSR

-- 80

--60 3 V

5 --40 >

-- 20

3 4 5 6 7 8 PH

Fig. 4. The effect of pH on volatile solids reduction (J%R) and soluble total organic carbon (TOC) in anaerobic sludge digestion; HRT 2 days (SRT 1.5-2 days) and 65°C.

S. Lee, J. Yu /Resources, Conservation and Recycling 19 (1997) 151-164 159

300 100

250 -- -- 80

3 200 --

-- 50 20 --

0 I I I I I I I I 0

0 0.5 1 1.5 HRT (hr)

2 2.5

Fig. 5. The effect of hydraulic retention time (HRT) on volatile solids reduction (VSR) and soluble total organic carbon (TOC) in anaerobic sludge digestion; pH 4 and 65°C.

compared in parallel (Fig. 7). PHB and sludge PHAs were degraded at a similar rate, 27 and 22% in 5 weeks, respectively.

Poly-3-hydroxybutyrate (PHB) in strong sulfuric acid solution was decomposed into crotonic acid, CH,-CHOH-CH,-COOH, the sole C, monomer while P(HB-co- HV), was decomposed into three components as shown in Fig. 8. Having three components in its hydrolysis solution, the sludge PHA is also a co-polymer (Fig. S), but contains mainly C, monomers (74%). This fact indicates that acetic acid, one of the major products from sludge digestion, was converted to C, monomers instead

Table 2 Production of PHAs by A. eutrophus in 48 h

Substrate Final cell g dry PHAs g/L PHAs/cell wt “/o TOC fed g/L TOC conver- mass/L sion %

HBu/HPr” 1.71 0.57 33.3 0.52 71.7 HBu/HPrb 1.35 0.79 58.5 0.26+0.26 81.5 Effluent’ 1.79 0.61 34.1 0.62 78.6

“50 wt % HPr, 50 wt %I HBr; 1.0 g acids/L was added at the beginning. %ame acids as “; half amount at the beginning, half at 24 h. “Supernatant of digested sludge (0.62 g TOC/L) was added at the beginning


3 6 7

Time (week) Fig. 6. Degradation of thermoplastics produced from the supernatant of digested sludge (sludge PHAs) in aerobic and anaerobic sludge suspensions at 2O”C, sea water and soil at room temperature (23°C).

of C, monomers during polymerization by A. eutrophus. It has been known that the presence of other monomers (C,, C,, C,) in PHAs affects considerably the physical properties of the polymers such as melting point, crystallinity, tensile and impact strength. The presence of C, monomer in P(HB-co-HV), for exam- ple, causes a lower melting point, but higher impact and tensile strength than PHB [8]. Fig. 9 shows the differential scanning calorimetry (DSC) thermograms of PHB, P(HB-co-HV) and sludge PHAs. The melting point of sludge PHAs is 167.5”C, a medium of the melting points of PHB (175.3”(Z) and P(HB-co-HV) (157.4”C), which implies the sludge thermoplastic has an average tensile and impact strength of PHB and P(HB-co-HV). The crystal structure usually affects the melting curve of polymers because crystallites can melt at different tempera- tures and adsorb different amount of heat. The similarity of the melting curves between PHB and sludge PHAs indicates that the sludge plastic has a simpler crystal structure than P(HB-co-HV). The lower heat flow (+ 65.7 J/g) required by sludge polymer melting than both commercial polymers may be attributed to its relatively lower crystallinity.

S. Lee, J. Yu / Resources, Conservation and Recycling 19 (1997) 1.5-164 161

4. Conclusion

About 50% of volatile solids in municipal sludge was digested under anaerobic thermophilic conditions, which means a 30% total solids reduction in typical municipal sludge containing 60% volatile solids. The volatile solids reduction was quite consistent and affected not very much by temperature, pH, HRT (SRT). However, pH and temperature had quite considerable effect on the formation of total soluble organic compounds. The carbon source of soluble organic compounds in the supernatant of digested sludge could be utilized by A. eutrophus to produce PHAs, and the efficiency was comparable to the carbon source of pure fatty acids. Both PHA contents in cells (33-34%) and TOC conversion in solution (70-80%) were similar for those two types of carbon source. The sludge PHA is a copolymer having mainly C, monomers (74%). Compared to PHB, the sludge PHA has a lower melting point, and hence better impact and tensile strength. Compared to P(HB-co-HV), the sludge PHA has a simpler melting curve shape, and hence more uniform crystal structure. The sludge thermoplastic can be degraded by more than 70% mass in 5 weeks in sludge suspensions, and has a similar biodegradability as

‘;;‘ 95 -- b

-s ; go -- E $ 85 -- Q 3 s 80 -- .- % uz 75 --

PHB does in soil.

100

0 1 2 3 4 5 6 Time (week)

Fig. 7. Degradation of commercial polyhydroxybutyrate (PHB) and thermoplastics produced from digested sludge (SPHAs) in soil at room temperature (23°C).

162 S. Lee, .I. Yu / Resources, Conservation and Recycling 19 (1997) 151-164

Fig. 8. GC chromatograms of hydrolyzed polyesters in 98% sulfuric acid solution; (A) polyhydroxybutyrate; (B) poly(hydroxybutyrate-co-hydroxyvalerate) with 8% PHV and (C) sludge PHA.

Acknowledgements

The research was supported by Environment and Conservation Fund of Hong Kong (ECWW94/95.EGOl). The authors thank Sha Tin Municipal Wastewater Treatment Center, Hong Kong, for sludge samples.


Fig. 9. Differential scanning calorimetry thermograms of (A) polyhydroxybutyrate, m.p. 175.3”C (B) sludge PHA, m.p. 167.5”C (C) poly(hydroxybutyrate-co-hydroxyvalerate) with 8”/0 PHV, m.p. 157.1”C.

164 S. Lee, J. Yu / Resources, Conservation and Recycling 19 (1997) 151-164

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Production of biodegradable thermoplastics from municipal sludge by a two-stage bioprocess