M.G.E. Albuquerque

Strategies for PHA production by mixed cultures and renewable waste materials.

Production of polyhydroxyalkanoates (PHA) by mixed cultures has been widely studied in the last decade. Storage of PHA by mixed microbial cultures occurs under transient conditions of carbon or oxygen availability, known respectively as aerobic dynamic feeding and anaerobic/aerobic process. In these processes, PHA-accumulating organisms, which are quite diverse in terms of phenotype, are selected by the dynamic operating conditions imposed to the reactor. The stability of these processes during long-time operation and the similarity of the polymer physical/chemical properties to the one produced by pure cultures were demonstrated. This process could be implemented at industrial scale, providing that some technological aspects are solved. This review summarizes the relevant research carried out with mixed cultures for PHA production, with main focus on the use of wastes or industrial surplus as feedstocks. Basic concepts, regarding the metabolism and microbiology, and technological approaches, with emphasis on the kind of feedstock and reactor operating conditions for culture selection and PHA accumulation, are described. Challenges for the process optimization are also discussed.

Polyhydroxyalkanoate (PHA) production by a mixed microbial culture using sugar molasses: Effect of the influent substrate concentration on culture selection

In Polyhydroxyalkanoate (PHA) production processes using Mixed Microbial Culture (MMC), the success of the culture selection step determines, to a great extent, the PHA accumulation performance obtained in the final PHA production stage. In this study, the effect of the influent substrate concentration (30-60Cmmol VFA/L) on the selection of a PHA-storing culture using a complex feedstock, fermented sugar molasses, was assessed. At 30 and 45Cmmol VFA/L, substrate concentration impacted on the process kinetics through a substrate dependent kinetic limitation effect. However, further increasing the carbon substrate concentration to 60Cmmol VFA/L, resulted in an unforeseen growth limitation effect associated with a micronutrient deficiency of the fermented feedstock (magnesium) and high operating pH. Struvite precipitation caused a nutrient limitation which prevented biomass concentration increase, thus causing the feast to famine length ratio to vary in the selection reactor, with subsequent impact on the selective pressure for PHA-storing organisms. A highly dynamic response of the selected population to transient conditions of feast to famine ratio, in the range of 0.21-1.1, was observed. Kinetic (limiting concentration of carbon source) and physiological (loss of internal growth limitation due to the shorter length of famine phase) effects, resulting from variation of the influent substrate concentration, were subsequently demonstrated in batch studies. The culture selected at an influent substrate concentration of 45Cmmol VFA/L showed the best PHA-storing capacity since neither substrate concentration nor feast to famine ratio were limiting factors. This culture, highly enriched in PHA-storing organisms (88%), reached a maximum PHA content of 74.6%.

Mixed culture polyhydroxyalkanoate (PHA) production from volatile fatty acid (VFA)-rich streams: effect of substrate composition and feeding regime on PHA productivity, composition and properties.

In this study, the possibility of manipulating biopolymer composition in mixed culture polyhydroxyalkanoate (PHA) production from fermented molasses was assessed by studying the effects of substrate volatile fatty acid (VFA) composition and feeding regime (pulse wise versus continuous). It was found that the use of a continuous feeding strategy rather than a pulse feeding strategy can not only help mitigate the process constraints of the pulse-feeding strategy (resulting in higher specific and volumetric productivities) but also be used as means to broaden the range of polymer structures. Continuous feeding increased the hydroxyvalerate content by 8% relatively to that obtained from the same feedstock using pulse wise feeding. Therefore, the feeding strategy can be used to manipulate polymer composition. Furthermore, the range of PHA compositions, copolymers of P(HB-co-HV) with HV fraction ranging from 15 to 39%, obtained subsequently resulted in different polymer properties. Increasing HV content resulted in a decrease of the average molecular weight, the glass transition and melting temperatures and also in a reduction in the crystallinity degree from a semi-crystalline material to an amorphous matrix.

Link between microbial composition and carbon substrate-uptake preferences in a PHA-storing community

The microbial community of a fermented molasses-fed sequencing batch reactor (SBR) operated under feast and famine conditions for production of polyhydroxyalkanoates (PHAs) was identified and quantified through a 16 S rRNA gene clone library and fluorescence in situ hybridization (FISH). The microbial enrichment was found to be composed of PHA-storing populations (84% of the microbial community), comprising members of the genera Azoarcus, Thauera and Paracoccus. The dominant PHA-storing populations ensured the high functional stability of the system (characterized by high PHA-storage efficiency, up to 60% PHA content). The fermented molasses contained primarily acetate, propionate, butyrate and valerate. The substrate preferences were determined by microautoradiography-FISH and differences in the substrate-uptake capabilities for the various probe-defined populations were found. The results showed that in the presence of multiple substrates, microbial populations specialized in different substrates were selected, thereby co-existing in the SBR by adapting to different niches. Azoarcus and Thauera, primarily consumed acetate and butyrate, respectively. Paracoccus consumed a broader range of substrates and had a higher cell-specific substrate uptake. The relative species composition and their substrate specialization were reflected in the substrate removal rates of different volatile fatty acids in the SBR reactor.

Mixed culture polyhydroxyalkanoates production from sugar molasses: The use of a 2-stage CSTR system for culture selection

Polyhydroxyalkanoates (PHAs) are promising biodegradable polymers. The use of mixed microbial cultures (MMC) and low cost feedstocks have a positive impact on the cost-effectiveness of the process. It has typically been carried out in Sequencing Batch Reactors (SBR). In this study, a 2-stage CSTR system (under Feast and Famine conditions) was used to effectively select for PHA-storing organisms using fermented molasses as feedstock. The effect of influent substrate concentration (60-120 Cmmol VFA/L) and HRT ratio between the reactors (0.2-0.5h/h) on the systems selection efficiency was assessed. It was shown that Feast reactor residual substrate concentration impacted on the selective pressure for PHA storage (due to substrate-dependent kinetic limitation). Moreover, a residual substrate concentration coming from the Feast to the Famine reactor did not jeopardize the physiological adaptation required for enhanced PHA storage. The culture reached a maximum PHA content of 61%. This success opens new perspectives to the use of wastewater treatment infrastructure for PHA production, thus valorizing either excess sludge or wastewaters.

Characterization of polyhydroxyalkanoates synthesized from microbial mixed cultures and of their nanobiocomposites with bacterial cellulose nanowhiskers.

The present work reports on the production and characterization of polyhydroxyalkanoates (PHAs) with different valerate contents, which were synthesized from microbial mixed cultures, and the subsequent development of nanocomposites incorporating bacterial cellulose nanowhiskers (BCNW) via solution casting processing. The characterization of the pure biopolyesters showed that the properties of PHAs may be strongly modified by varying the valerate ratio in the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymer, as expected. Increasing the valerate content was seen to greatly decrease the melting temperature and enthalpy of the material, as well as its rigidity and stiffness, resulting in a more ductile behaviour. Additionally, the higher valerate PHA displayed higher permeability to water and oxygen and higher moisture sensitivity. Subsequently, BCNW were incorporated into both PHA grades, achieving a high level of dispersion for a 1 wt.-% loading, whereas some agglomeration took place for 3 wt.-% BCNW. As evidenced by DSC analyses, BCNW presented a nucleating effect on the PHA matrices. BCNW also increased the thermal stability of the polymeric matrices when properly dispersed due to strong matrix-filler interactions. Barrier properties were seen to depend on relative humidity and improved at low nanofiller loadings and low relative humidity.

6.51 – Mixed Culture Processes for Polyhydroxyalkanoate Production from Agro-Industrial Surplus/Wastes as Feedstocks

Polyhydroxyalkanoates (PHAs) are a group of biologically synthesized polyesters that are considered promising eco-efficient bioplastics because they are both biobased and biodegradable, thus meeting the criteria of a closed loop life cycle. In the past decades, industrial biotechnology has devoted a considerable effort to PHA production by bacterial pure culture fermentation. However, PHAs have not yet entered bulk materials markets due to high production costs. The combined use of mixed microbial cultures (MMCs) and low-value feedstocks is currently under investigation in order to decrease operating costs. For the sake of enhanced PHA production, mixed cultures have to be preliminarily enriched in PHA-accumulating organisms. This is usually carried out under dynamic feeding of suitable carbon sources to create transient conditions of excess and lack of carbon availability, designated as feast and famine. PHA production is then usually carried out in a subsequent PHA accumulation stage. To make waste- and surplus-based feedstocks suitable for PHA production, acidogenic fermentation is used to convert their organic fraction into volatile fatty acids, which are viable precursors for mixed culture PHA synthesis. The main issues of the three-stage PHA production process from surplus-based feedstocks using MMCs are here described and discussed.

The relationship between mixed microbial culture composition and PHA production performance from fermented molasses

Polyhydroxyalkanoates (PHAs) are polyesters that can be produced from industrial wastewater or surplus products by mixed microbial cultures (MMC). To optimise PHA production by MMCs, the link between the microbial structure and function of these enrichments must be better established. This study investigates, for the first time, the impact of operational changes on the microbial community and the associated process performance of PHA producing MMCs. It was found that a PHA producing community fed with fermented molasses was dominated by a combination of Azoarcus, Thauera and Paracoccus, where the former two groups were present in highest abundance. Dominance of either Thauera or Azoarcus seemed to be determined by the organic loading rate imposed in the selection reactor. While higher Azoarcus enrichments led to higher PHA production yields and lower biomass growth yields as compared to Thauera, the Thauera abundance was strongly linked to higher hydroxyvalerate (HV) fractions. Paracoccus abundance was correlated with a lower PHA production capacity as compared to Azoarcus, and produced lower HV fractions than Thauera and Azoarcus. The findings of this study suggest that MMCs targeting the enrichment of Azoarcus as the primary biomass fraction with Thauera as a minor fraction lead to optimal specific PHA production and polymers with high HV content, which is likely to improve their mechanical properties.

Flux balance analysis of mixed microbial cultures: Application to the production of polyhydroxyalkanoates from complex mixtures of volatile fatty acids

Fermented agro-industrial wastes are potential low cost substrates for polyhydroxyalkanoates (PHA) production by mixed microbial cultures (MMC). The use of complex substrates has however profound implications in the PHA metabolism. In this paper we investigate PHA accumulation using a lumped metabolic model that describes PHA storage from arbitrary mixtures of volatile fatty acids (VFA). Experiments were conducted using synthetic and complex VFA mixtures obtained from the fermentation of sugar cane molasses. Metabolic flux analysis (MFA) and flux balance analysis (FBA) were performed at different stages of culture enrichment in order to investigate the effect of VFA composition and time of enrichment in PHA storage efficiency. Substrate uptake and PHA storage fluxes increased over enrichment time by 70% and 73%, respectively. MFA calculations show that higher PHA storage fluxes are associated to an increase in the uptake of VFA with even number of carbon atoms and a more effective synthesis of hydroxyvalerate (HV) precursors from VFA with odd number of carbons. Furthermore, FBA shows that the key metabolic objective of a MMC subjected to the feast and famine regimen is the minimization of the tricarboxylic acid cycle fluxes. The PHA flux and biopolymer composition (hydroxybutyrate (HB): HV) could be accurately predicted in several independent experiments.

The impact of pH control on the volumetric productivity of mixed culture PHA production from fermented molasses

Polyhydroxyalkanoate (PHA) production via mixed microbial cultures (MMCs) can potentially decrease process operational costs as compared to conventional pure culture techniques. However, the volumetric productivity of PHA by MMCs must be augmented to increase its cost competitiveness. For this purpose, a three‐stage bioreactor system was operated in this study, with (i) anaerobic fermentation of molasses, (ii) culture selection, and (iii) PHA accumulation and harvesting stages. In stage 2, bioreactor operation with pH control at 8 led to twice the biomass concentration (up to 8 g VSS L−1, where VSS is the volatile suspended solids) as compared to operation without pH control (maximum pH 9). No loss in the specific PHA storage efficiency was observed (PHA content up to 57.5% and PHA storage rate up to 0.27 Cmol PHA Cmol X−1 h−1, where X is the active biomass), thereby resulting in twice the volumetric PHA production rate. The limited biomass growth at the higher pH level was not due to nutrient limitation, but likely to a shift in the microbial community. It is hypothesized that the increased enrichment of Azoarcus at pH 8 led to higher PHA productivity. pH control in the culture selection stage can lead to enhanced PHA production from MMCs, improving the viability of the process.