Production of Biodegradable Plastics (Polyhydroxyalkanoate - PHA) through Biological Processes: Challenges and Opportunities Tjandra Setiadi, Rety Setiawaty and Martha Aznury
Presented at International Postgraduate Conference on Biotechnology, August 25, 2016, ITS, Surabaya
Department of Chemical Engineering ITB
[email protected] 1
Outline Introduction Polyhidroxyalkanoate (PHA) Biosynthesis of PHA PHA Production by Mixed Culture PHA Production by Pure Culture Challenges and Opportunities
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Introduction
degradable plastic, n—a plastic designed to undergo a significant change in its chemical structure under specific environmental conditions resulting in a loss of some properties that may vary as measured by standard test methods appropriate to the plastic and the application in a period of time that determines its classification.
biodegradable plastic, n—a degradable plastic in which the degradation results from the action of naturally-occurring micro-organisms such as bacteria, fungi and algae. Definition according ASTM and ISO
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World’s Biodegradable Plastic Demand 2010
2011
2016
(Million Pound)
(Million Pound)
(Million Pound)
PLA1
470
578
1.655
23.4
Starch-based
251
289
582
15.0
PHA & other polyesters2
50
65
279
33.8
Total
771
932
2516
22.0
Biodegradable Polymer/ Plastic
Average annual increase (%)
Source : Plastics Europe Market Research Group (PEMRG), BCC-Research
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Biodegradable plastics
Partially biodegradable
Completely biodegradable
PCL
Starch/PE
Natural
Fermentation
PHA
Modification of cellulose products
Cellulose
Starch/ PVA
Fermentation/synthesis
Polilactide
Starch/ PCL
Chemical synthesis
Aliphatic polyester PET-PCL
Chitosan/ cellulose
Biodegradable plastics (Chang, 1994) PE – polyethylene; PCL – polycaprolactone; PHA – polyhydroxyalkanoate; PET - Polyethylene5 terephthalate; PVA - polyvinyl alcohol
Polyester Polymers
Polyesters
Aromatic (Ring Polymers)
PBS (butyl succinate)
PCL (polycaprolactone)
PBSA (Polybutylene succinate adipate)
PHA (polyhydroxyalkanoates)
PHB (polyhydroxybutyrate)
PHB/PHV
Aliphatic (linear Polymers)
PHB/PHH
PLA (polylactic acid)
PHV (polyhydroxyvalerate)
PHH (polyhydroxyhexanote)
PHB/PHV
PHB/PHH
Modified PET
AAC (aromatic copolyesters)
PBAT (polybutylene adipate terphthalate)
PTMAT (polymethylene adipate terephthalate
Synthetic / Renewable Synthetic / Nonrenewable Natural / Renewable
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Polihydroxyalkanoate (PHA)
Poly Hydroxy Alkanoate
Polyhydroxyalkanoates (PHAs) are polyesters of various (R)-hydroxy carboxylic acids, which are accumulated as carbon/energy storage or reducing power materials in numerous microorganisms usually under the unfavourable growth condition in the presence of excess carbon source 7
Polyhydroxyalkanoate (PHA)
Ojumu et al., 2004 8
PHA advantages • complete biodegradability • wide ranging physical and mechanical properties after co-polymerization • biocompatibility to human tissue in surgical applications
PHA Biosynthesis
Source: Magdouli et al., 2015
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PHA Biosynthesis • PHA is biodegradable and can be produced biologically using various substrates including organic wastewater (Du et al., 2001). This makes PHA an alternative to petroleum-based plastics used today. • Currently, the commercial PHA branded with different trade names, such as Biopol, Metabolix, Nodax, etc. produced from glucose using different species (Magdouli et al., 2015) 11
Commercialized PHAs with their trade names
Polymer
Strain
P(3HB) PHB and PHBV
Manufacturers
Rhizobium
W.R. Grace and Company
Cupriavidus necator NCIB 11599
ICI Ltd (Imperial Chemical Industries Ltd) (BiopolTM)
Recombinant E. coli K12
Metabolix, Inc.
Recombinant E. coli
(NodaxTM)
Table 1 Commercialized PHAs with their trade names and manufacturing companies. -
PHB and mclPHA P(3HB-co3HHx)
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PHA Biosynthesis • It was commercially produced as early as 1976 by ICI, however the production was terminated in 1998 for a number of reasons (Koller et al., 2016). • Recently, on July 6, 2016, Newlight Technology (USA) and Paques Holding bv (the Netherlands) have come to an agreement that will allow Paques to manufacture, process and sell bioplastics (PHA) based on Newlight’s biocatalyst process to convert greenhouse gases (methane) to PHA (named AirCarbon) at a rate of up to 1.3 million metric tons/year 13
PHA Production • However, the drawback is that the PHA production cost is still much higher than that of petroleum plastics, due to the cost of the substrate (Bengtsson et al., 2008). • On the other hand to use the pure culture also involves the high operational costs due to media sterilization and reactor maintenance (Reddy and Mohan, 2012a). • The potential reduction in cost is using organic material from agriculture and food industry wastewater as much as possible and mixed cultures system as the microbial agent (Din et al., 2012; Reddy and Mohan, 2012b).
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PHA Production with Mixed Cultures Setyawati, R., Setiadi, T., Katayama-Hirayama, K., Kaneko, H. and Hirayama, K. (2012). Polyhydroxyalkanoate (PHA) production from tapioca industrial wastewater treatment: Influence of operating conditions on PHA content, Sustain. Environ. Res., 22 (2), 123-127
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Activated Sludge and Wastewater
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Sequencing batch reactor (SBR) • fill and draw processes : fill, react, settle/sedimentation, draw/decant, and idle • aeration and settling in the same tank • can manipulate the environmental change for microorganisms activities
Sequencing batch reactor (SBR)
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Conclusions • Although results showed large variations, the following conclusions can be drawn: • (1) Tapioca industrial wastewater treatment using a SBR with activated sludge as the source of microorganisms can produce PHA with average PHA content 0.03-0.10 g PHA/g MLSS. • (2) COD was not effectively removed. Average COD removal efficiencies were 20-40%. And • (3) Combination of aerobic and anaerobic periods did not have a significant effect on PHA content and COD removal.
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PHA Production with a pure culture Setiadi, T., Aznury, M., Trianto, A. and Pancoro, A. (2015). Production of polyhdroxyalkanoate (PHA) by Ralstonia eutropha JMP 134 with VFAs from palm oil mill effluent as precursors. Water Science & Technology, 72(11), 1889-1895.
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Materials and Method (1) Acidogenic fermentation
Distillation POME
Anaerobic fermentation
Fermented POME
Mineral medium
Figure 1. Two-step PHA production process from POME by Ralstonia eutropha JMP 134
Ralstonia eutropha Glucose
VFAs
Aerobic fed-batch fermentation
PHA
(2) PHA production
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The Schematic diagram of PHA production Cell Cell production
Substrate
PHA production
VFA from POME
Separation
Supernatant (effluent)
Cell destruction
PHAs Purification
Addition of VFA either in Batch or Fed-Batch Mode
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Identification of PHA with different analysis methods FTIR Analysis Precursor
Cultivated Mode
VFAs from Distillated Fermented POME (DFP)
Batch
VFAs from Distillated Fermented POME (DFP)
Fed-Batch
Melting Point (oC)
Wave length (cm-1)
Possible structure ofPHA
GC Analysis Monomer compostion (%mol)
169.5
1.728 1.282
3-HB
100(3-HB)
139
2.927 1.737 1.303 1.229 1.196 797
3-HB and 3-HV
88(3-HB), 12(3-HV)
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H NMR analysis
References Zakaria, et al. 2010:Chakrabor ty et al. 2009 (GC) Choi dan Lee, 1999 (GC) Shamalaet al. 2014 (FTIR and 1 H NMR)
It could be determined that the PHA from batch mode was fully mono polymer of 3-hydroxybutyrate (100 % 3HB) and from fed-batch mode was a copolymer with 3-hydroxyvalerate content of 12 % mol (% 3-HV). 23
Comparison of the batch and fed-batch mode operation in PHA production Batch Substrate/ microorganism
VFAs
Fed batch
Productivity (g/L/h)
PHA (g/gDCW)
3HV (% mol)
0.014
39
0
Glucose/ Ralstonia eutropha JMP 134
VFAs from Distillated Fermented POME (DFP)
Mineral salts medium (MSM)/ Comamonas sp. EB172
VFAs (fermented POME)
Glucose/ Ralstonia eutropha JMP 134
VFAs from Distillated Fermented POME (DFP
Mineral salts medium (MSM)/ Comamonas sp. EB172
acetic, propionic acids
-
20
9
Condensed corn solubles/ Ralstonia eutropha
acetic acid
0.024
29.3
-
Substrate/ microorganism
VFAs
References
Productivity (g/L/h)
PHA (g/gDCW)
3HV (% mol)
0.048
51
12
This study
-
49
12
Zakaria et al..(2010)
Chakraborty et al.(2009)
Glucose/ Ralstonia eutropha
VFAs (fermented POME)
0.01
51
-
Hassan et al.(2002)
The PHA productivity in the fed-batch mode was 0.048 g/L/h, it is much higher than those of other studies (Table 3) and it was not much different to the productivity obtained by Campanari et al. (2014), i.e. 0.06 g/L/h, using VFAs from fermented Olive Oil Mill wastewater in a mixed culture. However it is in the lower range of values obtained by pure cultures reported, namely 0.04 to 0.9 g/L/h (Kim, 2000) 24
Conclusions • The highest VFAs production from POME was obtained at an anaerobic fermentation time of 1 day and pH of 4.43 with the distillated fermented-POME (DFP) consisted of acetic, propionic and butyric acid having a concentration of 2.79 g/L, 1.18 g/L, and 3.04 g/L, respectively. • PHA productivity in the fed batch mode gave a value of 0.048 g/L/h being an increase of 342 % compared to the batch mode. The addition of DFP in a fed batch mode could also increase the 3HV became 12 % mol. 3HV, whereas in the batch mode was only 0% mol. 3HV. • The PHA product structure could be well identified by different analysis methods, such as melting point, GC, FTIR, and NMR. These methods supported each other resulting the products were composed of 3-HB and 3-HV functional group. 25
Examples of PHA Products
Challenges and Opportunities • Although there is an increase research on this topic in the last twenty years, however the industrialization of this product is still a challenge. • The sustainability of PHA production in the near future will be depend on several factors, such as strain selection, feedstock selection, bioreactor cultivation mode, downstream processing and product processing development.
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Challenges and Opportunities
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Koller, M. Marsalek, L. de Sousa Dias, M.M., Braunegg, G. (2016)
Production Strain Selection • Wild-type vs genetically engineered strains • Mesophile vs extremophile organisms • Pure culture vs mixed microbial culture
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Feedstock Selection • Ethically clear (no interference with food- and feedprocesses • Seasonal availability of feedstocks (“Off-season” availability) • Addressing variability and stability of feedstock composition • Impact of applied feedstock on polymer composition (homo- vs co-polyester); quality (molar masses, coloration, etc.) and sensory quality (coloration, odor) • Upgrading of waste streams to feedstocks • Integration into existing production lines, etc. 34
Bioreactor Cultivation Mode (“Process Design”) • Discontinuous vs single- or multistage continuous mode • Solid state fermentation • Recycling of Spent fermentation broth • Self-sufficiency of energy supply, etc.
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Downstream Processing • Solvent selection (non-toxic solvents, recyclability) • Solvent-free methods • Recycling and reuse of cell debris and other side streams of recovery process, etc.
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Processing • Production of compatible Composites and Blends by utilization of ecologically benign materials (preferably inexpensive and renewable), etc.
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West Hall INSTITUT TEKNOLOGI BANDUNG
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