Ecoengineering of a Mixed Microbial Culture to maximize

Eco-engineering of a Mixed Microbial Culture to maximize PHA production from a surplus feedstock: sugar molasses Maria A. M. Reis CQFB-Requimte, FCT-UNL, Portugal

> Motivation > 1. Use of waste-based feedstocks for bioproduction of added value products Effluent/waste/ surplus Decanting Biological Reactor Treated Effluent Sludge treatment and disposal High Value product

> Motivation > 2. Sustainable bioplastic production: biobased and biodegradable Derived from oil limited reserves Synthetic plastics ~200 million ton/year millions of tons of waste recalcitrant to biological degradation Biobased+Biodegradable (B 2 B) plastics Biological synthesis Renewable Feedstock Biodegradation Biomass <20% recycled or incinerated >80% landfills or marine environments

> Motivation > 3. Polyhydroxyalkanoates (PHA) • Biologically synthesised polymers • Linear polyesters • Bio-based sustainable process • Biodegradable • Biocompatible High replacement • Thermoplastics potential vs polyolefins • High molecular weights (good thermoplastic ppties) • 100% water resistant H O C R O (CH 2)n C 100 - 30000 2 types of PHA: • scl-PHA if R up to = CH 3 C 3 (similar to polypropilene) poly-3 -hydroxybutyrate PHB R = C 2 H 5 poly-3 -hydroxyvalerate PHV • mcl-PHA if R = C 4 – C 9 (similar to low density polyethylene) PHA properties depend on PHA R composition (type and fraction of different HA) Co-polymers present improved mechanical and thermal properties. eg. P(HB-co-HV) more pliable than PHB

> Motivation > 4. Pure Cultures versus Mixed Cultures Substrate costs Upstream investment costs Operational costs Product yield on substrate Cellular PHA content Downstream costs Product properties Industrial tradition Mixed Pure

> Motivation > 5. Enrichment of Mixed Cultures in PHA storing organisms Aerobic Dynamic Feeding (ADF) or “Feast and Famine” 0. 4 0. 08 Famine 0. 3 0. 06 VFA PHA 0. 2 0. 04 miu 0. 1 0. 02 0 0. 00 0 2 Long famine Pulse feeding of excess phase carbon substrate Internal growth limitation miu (h-1) f. VFA, f. PHA (Cmol/Cmol) Feast Physiological adaptation 4 6 Time (h) 8 Stored PHA used as energy and carbon source 10 12 Competitive advantage

Aim 1. Understand the mechanisms of Feast and Famine Process 2. Selection of reactor configuration 3. Development of a metabolic model 4. Manipulation of the polymer properties

> 1. Optimisation of selection efficiency in SBR (2) Culture selection stage Cane molasses MF membrane Anaerobic CSTR Fermented molasses (1) Fermentation of feedstock Clarified fermented molasses SBR Biomass Sludge Batch reactor (3) PHA production stage PHA Extraction and purification

> 1. Optimisation of selection efficiency in Sequencing Batch Reactor (SBR) SBR cycle of operation Fill Feast/Famine TOC VFA PHB PHV Ac Prop But Val NH 4 5 4 60 3 40 2 20 NH 4 (Nmmol/L) TOC, VFA, PHA (Cmmol/L) 80 Settling Draw 1 0 0 0 2 4 6 8 10 12 Time (h) SBR cycle of operation

> 1. Optimization of selection efficiency in SBR 1 Role of Influent Substrate Concentration on the SBR F/F Ystorage Ygrowth Storage versus growth performance over time in SBR operated at different influent substrate conc. (30, 60 and 45 Cmmol VFA/L).

> 1. Optimization of selection efficiency in SBR 1 Effect of feast and famine ratio on the PHA storage vs growth Immediate physiological effect: longer famine increase of growth limitation due to physiological adaptation Carbon preferably driven toward PHA storage On the long run: Lower YX/PHA during feast higher N during famine increased competitive advantage of PHA-accumulating organisms higher enrichment in PHA storing organisms

> 1. Optimization of selection efficiency in SBR Effect of SBR selection efficiency on subsequent batch PHA production • 78% PHA content • YPHA/VFA = 0. 81 Cmol PHA/Cmol VFA • q. P = 0. 43 Cmol PHA/Cmol X. h SBR_30 SBR_60 SBR_45 PHA content (%) 80 70 60 50 40 30 20 10 0 0 4 8 12 Time (h) 16 20 24

> 2. Selection of Reactor configuration Continuous same configuration (2)Mode: Culture selection stage as WWTP Return sludge Cane molasses MF membrane Anaerobic CSTR Fermented molasses (1) Substrate production stage Clarified fermented molasses 1 Aerobic Feast Biomass Batch reactor Biomass (3) PHA production stage Aerobic Famine Settler

> 2. Selection of Reactor configuration Effect of HRT ratio and substrate concentration on selection efficiency

> 2. Selection of Reactor configuration SBR versus 2 -stage continuous system: different substrate concentration profiles

> 3. Development of a metabolic model • Model for a generic VFA uptake • Includes intracellular regulation factor (similar approach to Venkatesh et al 1997)

> 3. Metabolic model validation First model for PHA production from complex feedstocks Adjusts the experimental data very well can be used to optimize culture selection and PHA production stage

M > 4. Manipulation of polymer composition and properties a n i p u l a t i o n o f p o l y m e r ct o h rm p o Effect of VFA profile (HAc/HProp/ HBut/HVal) 60/16/20/04 32/19/28/21 Effect of Feeding Regime Pulse-Feeding Continuous Feeding P(HB-co-HV) 80: 20 70: 30 P(HB-co-HV) 69: 31 61: 39

> 4. Manipulation of polymer composition and properties • High molecular weight: Mw of 2. 2 – 6. 5 x 105; with PDI of 2. 3 – 2. 7 • Tm and Tg dependent on HV content, with constant Tmax enabling wide processing window (Tm 140 ºC and Tmax 240 ºC). • Similar variation between thermal properpties and HV content as reported for other mixed and pure cultures.

Carbon COD Balance WWTP/Biorefinery: WWTP Effluent treatment + PHA production Effluent treatment 70% 50% PHA Sludge + O 2 COD + O 2 50% COD CO 2 15 -20% Sludge + O 2 10 -15% CO 2

> Main Achievements • Undestanding of the Feast and famine mecahnisms allow to select a culture (88% PHA storing organisms) with high performance (78% PHA content); • A continuous feast and famine system (similar to a WWTP configuration) was succefully operated confirming the potential use of WWTP facilties for PHA production; • A metabolic model developed for complex feedstocks and mixed cultures which can be used for process optimization; • Control the co-polymer composition and thus polymer properties by manipulation of the VFA profile (anaerobic fermentation conditions, eg. p. H) and/or through the feeding regimen of the accumulation stage; • The co-polymers produced showed similar properties to co-polymers produced by pure cultures, enabling for wide processing windows.

> Ackowledgments Research team Maria A. Reis- REQUIMTE Graça Albuquerque- REQUIMTE Rui Oliveira- REQUIMTE João Dias-REQUIMTE Filipa Pardelha- REQUIMTE Eric Pollet- Univ. Strasburg > Ackowledgments Acknowledgments FCT – Fundação para a Ciência e Tecnologia “Sustainable Microbial and Biocatalytic Production of Advanced Functional Materials”, EU Integrated Project, Contract nº 026515 -2; 2006 -2008. Refinaria de Açúcares Reúnida (RAR), Portugal