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# Determination of the extraction kinetics for the quantification of polyhydroxyalkanoate monomers in mixed microbial systems - ScienceDirect

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Process Biochemistry
Volume 48, Issue 11, November 2013, Pages 1626-1634
Determination of the extraction kinetics for the quantification of polyhydroxyalkanoate monomers in mixed microbial systems
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Ana B. Lanham a
, 
Ana Rita Ricardo a
, 
Maria G.E. Albuquerque a
, 
Filipa Pardelha a
, 
Mónica Carvalheira a
, 
Marta Coma b
, 
Joana Fradinho a
, Gilda Carvalho a, Adrian Oehmen a
, Maria A.M. Reis a
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https://doi.org/10.1016/j.procbio.2013.07.023
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Highlights
•
Comprehensive analysis of factors affecting the extraction of PHA monomers.
•
HA extraction differed between floccular and granular mixed cultures.
•
Larger and more substituted monomers have slower extraction kinetics.
•
Modified HA extraction procedures are proposed for different systems.
•
HA extraction impacts the assessment of PHA biopolymer quantity and quality.
Abstract
For the first time, a systematic approach was conducted to determine the key factors influencing the kinetics of hydroxyalkanote (HA) extraction in biological systems. Six mixed microbial systems where polyhydroxyalkanoate (PHA) is produced were evaluated. Experiments were carried out for full-scale and lab-scale activated sludge systems using different configurations (containing floccular or granular sludge), as well as specific PHA accumulating cultures that contain high or low intracellular PHA fractions. The overall reaction was limited by the kinetics of the PHA hydrolysis in floccular cultures, whereas in granular cultures, it was limited by the cell lysis step. The monomeric composition of the polymer also had an impact on the HA extraction rate: higher acid concentration and a longer digestion time should be employed when cells accumulate monomers with more substituents, such as hydroxy-2-methylbutyrate (H2MB) and hydroxy-2-methylvalerate (H2MV). This study optimised the method for HA extraction, which impacts the assessment of the quantity and quality of PHA biopolymers.
Introduction
Polyhydroxyalkanoate (PHA) are polymers composed by a family of polyester monomers which include over 110 different molecules with varying backbone length from 4 to 16 carbons [1]. PHA synthesis from an external carbon source has been identified in a great number of different bacteria (e.g. Ralstonia eutropha, Azotobacter vinelandii, Candidatus Accumulibacter phosphatis), which utilise this polymer as a carbon, energy and reducing equivalents storage product when facing limiting conditions (e.g. nutrients or oxygen) [2], [3]. PHA is accumulated inside the bacterial cells as discrete granules that are often composed of different monomers, forming a co-polymer, e.g., poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (co-PHB-PHV).
PHA is a biodegradable and renewable alternative to the fossil-produced polyesters that currently hold the majority of the plastics market. Using pure or mixed cultures fed on waste material or industrial by-products, several authors were able to show the feasibility of producing a new generation of more sustainable and biocompatible plastics [4], [5], [6].
Additionally, PHA also plays a significant role as a storage polymer in environmentally engineered processes [3], where various organisms rely on PHA in order to survive in these systems. Therefore, PHA is included in biological wastewater treatment models [7] and is often a key compound when addressing the metabolism of bacteria present in such systems [8], [9].
While PHA is a highly important biopolymer to both the bioplastics and wastewater treatment industries, very little focus has been paid to the extraction step necessary to recover hydroxyalkanoate (HA), particularly from mixed microbial cultures [4]. This is of particular importance for the quantification of the individual monomers, which have a high impact on the mechanical properties of the polymer, and furthermore impact the determination of the kinetic and stoichiometric parameters associated with mathematical models. The method that is most widely used for HA extraction involves an acidic methanolysis reaction, which was initially developed by Braunegg et al. [10] for a PHB producing pure culture, and later adapted for HV and medium-chain length (MCL) monomers [11], [12], [13]. In mixed microbial cultures, microorganisms are often not in suspension but form complex and heterogeneous structures, such as flocs or granules, aggregated due to extracellular polymeric substances. The biomass properties are likely to affect HA recovery, where the effect of e.g. higher cell packing in the case of granules was also found to impact glycogen extraction [14]. Nevertheless, the factors affecting HA extraction kinetics have never been studied before.
This work used a comparative approach in different microbial systems to systematise the factors impacting HA extraction. For the first time, a systematic approach using design of experiments was conducted for six mixed microbial cultures from different processes, with different microbial characteristics producing the most common HA monomers found in these systems (HB, hydroxy-2-methylbutyrate (H2MB), HV and hydroxy-2-methylvalerate (H2MV)). These cultures included PHA accumulating organisms (higher PHA content), activated sludge (lower PHA content) as well as floccular and granular systems. The determination of the impact of these factors on different microbial systems accumulating various co-polymers is a valuable tool to guide researchers in characterising the quantity and quality of the PHA in their specific system.
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Section snippets
Microbial cultures tested
Six different mixed microbial cultures were selected for this study, which displayed different PHA accumulation capacities and compositions. These cultures result from ongoing work at the authors’ laboratories.
The first and the second microbial cultures tested (MC1 and MC2) were from the same PHA producing culture, maintained in a 800 mL sequencing batch reactor (SBR), run in aerobic dynamic feeding conditions and fed with fermented sugar cane molasses [15]. MC1 was obtained through a PHA
The effect of monomeric composition and sludge structure on the HA extraction rate
The impact of the acid concentration on extraction kinetics was assessed by quantifying the different HA monomers at increasing digestion times. Two parallel hydrolysis curves were obtained using acidic methanol with 3 and 20% sulphuric acid (Fig. 1) and approximately 3 mg/mL of biomass. The GC peak areas were normalised with the internal standard and the biomass weighed for each sample.
The extraction rates increased from 2 to 4 fold when using 20% acidic methanol instead of 3%. For example, the 
The overall effect of each parameter on the extraction and quantification of PHA monomers
Acid concentration, time of digestion and biomass concentration had an effect on the HA extraction rate and recovery. The results from the hydrolysis profiles, performed on only some cultures, were consistent with the results from the DOE.
The biomass concentration limited the recovered fraction of HB and HV in values lower than 3 mg/mL, which was supported by the DOE results since they indicated that in the 2–8 mg/mL tested range, biomass concentration had a positive effect, although only a
Conclusions
This study systematises, for the first time, the factors determining the efficiency of HA extraction using acidic methanolysis. Floccular or granular cultures should be treated differently, since the HA recovery is limited by the hydrolysis kinetics in the former and the cell lysis step in the latter. Hydrolysis kinetics decrease with an increase in the size and number of substituent groups of the co-polymer, due to steric hindrance mechanisms, an effect that can be counterbalanced with an
Acknowledgments
The authors wish to thankfully acknowledge funding from the Fundação para a Ciência e Tecnologia through the PhD grants SFRH/BD/25275/2005, SFRH/BD29477/2006, SFRH/BD38763/2007, SFRH/BD42085/2007, SFRH/BD65113/2009, the Post-Doc grants SFRH/BPD/88382/2012, SFRH/BPD/70185/2010 and the research projects PTDC/EBB-EBI/103147/2008 and PEst-C/EQB/LA0006/2011. Marta Coma also wishes to thank the financial support of the Spanish Government through the research project MICINN-PT2009-0047 and the 
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