DEPARTAMENT OF CHEMISTRY
Use of bio-waste for polyhydroxyalkanoates (PHA) biosynthesis: enhanced accumulation towards a sustainable and economical bio- process
FERNANDO RAMOS SILVA Master’s in biotechnology
DOCTORATE IN CHEMICAL AND BIOCHEMICAL ENGINEERING
DEPARTAMENT OF CHEMISTRY
Use of bio-waste for polyhydroxyalkanoates (PHA) biosynthesis: enhanced accumulation towards a sustainable and economical bio- process
FERNANDO RAMOS SILVA Master’s in Biotechnology
DOCTORATE IN CHEMICAL AND BIOCHEMICAL ENGINEERING
Adviser: Maria D’Ascensão Carvalho Miranda Reis
Full Professor, NOVA University Lisbon, Portugal
Co-adviser: Gilda de Sousa Carvalho Oehmen
Senior Lecturer, The University of Queensland, Australia
Examination Committee:
Chair: João Paulo Serejo Goulão Crespo, Full Professor, NOVA University Lisbon, Portugal
Rapporteurs: Bruno Sommer Ferreira, Chief Executive Office, Bi- otrend SA
Mauro Majone, Full Professor, Sapienza University of Rome, Italy
Adviser: Maria D’Ascensão Carvalho Miranda Reis, Full Professor, NOVA University Lisbon, Portugal Members: Cláudia Filipa Reis Galinha Loureiro, Senior Re-
searcher, NOVA University Lisbon, Portugal Francesco Valentino, Assistant Professor, Ca’ Fos- cari University of Venice, Italy
DEPARTAMENT OF CHEMISTRY
Use of bio-waste for polyhydroxyalkanoates (PHA) biosyn- thesis: enhanced accumulation towards a sustainable and economical bioprocess
FERNANDO RAMOS SILVA
Master’s in Biotechnology
Use of bio-waste for polyhydroxyalkanoates (PHA) bio-synthesis: enhanced accumulation to- wards a sustainable and economical bioprocess
Copyright © Fernando Ramos Silva, NOVA School of Science and Technology, NOVA University Lisbon.
The NOVA School of Science and Technology and the NOVA University Lisbon have the right, perpetual and without geographical boundaries, to file and publish this dissertation through printed copies reproduced on paper or on digital form, or by any other means known or that may be invented, and to disseminate through scientific repositories and admit its copying and distribution for non-commercial, educational or research purposes, as long as credit is given to the author and editor.
A CKNOWLEDGMENTS
A experiência deste doutoramento foi sem dúvida única e memorável, que me permitiu crescer e aprender imenso. Como tal, quero agradecer a todas as pessoas que, das mais vari- adas formas, contribuíram para que isso acontecesse.
Em primeiro lugar, gostaria de agradecer às minhas orientadoras Professora Doutora Maria Ascensão Reis e Professora Doutora Gilda Carvalho, por esta incrível oportunidade e pelo apoio que me deram ao longo do doutoramento. Quero reforçar o agradecimento à Pro- fessora Ascensão Reis, pelas condições de trabalho que me proporcionou ao longo destes anos, e por me ter permitido colaborar nos inúmeros projetos de investigação em que cola- borei ao longo destes anos.
Quero também agradecer aos meus colegas do grupo BioEng, em especial a todos aque- les com quem tive a sorte de trabalhar na piloto, e aos meus alunos de mestrado, com quem trabalhei lado a lado durante este percurso. Foi um prazer fazer ciência com vocês!
Agradeço também o apoio financeiro da Fundação para a Ciência e Tecnologia através da bolsa de doutoramento PD/BD/126626/2016 e às instituições que me acolheram FCT/UNL e UCIBIO.
Gostaria também de deixar um grande obrigado a todos os meus amigos que me acom- panharam ao longo deste percurso… Sem dúvida que a vossa presença ajudou a manter uma perspetiva mais leve!
Por último, um muito obrigado aos meus pais e namorada, a quem dedico esta tese. Sou eternamente grato pela vossa existência, amor e imensurável apoio. Obrigada por estarem sempre presentes… Com vocês as coisas tornam-se sempre mais fáceis.
Obrigada a todos.
“If you can believe in something and put it in your mind and heart, it can be realized.” (Eliud Kipchoge).
A BSTRACT
Polyhydroxyalkanoates (PHA) are considered a promising alternative to oil-based plas- tics. They are naturally occurring polyesters, biocompatible and biodegradable into CO2 and H2O. Nowadays, PHA are produced through a costly process using single cultures at full-scale, which has prevented the widespread use of this plastics for cheaper applications.
An alternative bioprocess with mixed microbial cultures (MMC) has been considered as an alternative approach using wastes as feedstock. This process generally consists of acido- genic fermentation of a waste, selection of a PHA-accumulating culture and PHA accumulation.
This thesis has mostly focused in the last two steps, particularly on overcoming the complexity of fermented wastes by valorizing them into PHA with a focus pilot-scale experiments.
In the first experimental study, the typical 3-stage process was setup at pilot scale. A caproate-rich fermentate was produced from fruit waste and used in the subsequent stages to produce a 3-hydroxyhexanoate-rich PHA, containing up to 66% (wt.) of this medium chain length (mcl) monomer. The metabolism of caproate was further elucidated and a metabolic pathway to produce this mcl monomer was proposed.
A second study was carried out using a nitrogen-rich fermented food waste as substrate for bench-scale and pilot-scale PHA processes, which resulted in the production of a 3-hy- droxyoctanoate-containing tetra-polymer. A mass balance was performed to understand the fermentation products' metabolism and a projection to latent structures (PLS) model was cre- ated to predict PHA production.
In the third study, ammonia was stripped out from fermented sewage sludge and fed to a bench-scale selection reactor during the famine phase as nitrogen source in a partially un- coupled carbon and nitrogen feeding strategy. This additional step resulted in an increase in 77% of productivity compared to similar operating conditions without nitrogen removal be- forehand.
The impact of using a nitrogen-rich fermented food waste as nitrogen source in the fam- ine phase, while a fermented fruit waste was used as carbon source in the feast phase was evaluated in the fourth study. Although a slight decrease in PHA content and productivity was observed, the change in operating conditions allowed the use of a low-cost waste instead of a supplementation of nutrients.
This Thesis demonstrates how to overcome process challenges through process adapta- tions to produce an added-value product from highly abundant wastes.
Keywords: polyhydroxyalkanoates, mixed microbial cultures, fermented waste, medium chain length monomers, nitrogen
R ESUMO
Polihidroxialcanoatos (PHA) são considerados uma alternativa promissora aos plásticos derivados do petróleo. Os PHA são poliésteres naturais, biocompatíveis e biodegradáveis em CO2 e H2O. Hoje em dia, os PHA são produzidos através de um processo caro usando culturas puras à escala industrial, o que tem impedido um uso mais abrangente destes plásticos em aplicações de baixo custo.
Um bioprocesso alternativo usando culturas microbianas mistas (MMC) tem sido consi- derado com alternativa usando resíduos como substratos. Este processo consiste geralmente na fermentação acidogénica do resíduo, seleção da cultura bacteriana acumuladora de PHA e a produção de PHA. Esta tese foca-se nos últimos dois passos, particularmente em ultrapassar as barreiras que os resíduos fermentados possuem, valorizando-os a PHA em experiências à escala piloto.
No primeiro estudo experimental, o processo convencional de três passos foi implemen- tado à escala piloto. Um efluente rico em caproato foi produzido a partir de resíduo de fruta e usado nos passos subsequentes para produzir um PHA rico em 3-hidroxihexanoato, contendo até 66% (wt.) deste monómero de cadeia média (mcl). O metabolismo do caproato foi eluci- dado e a via metabólica da produção deste monómero mcl foi proposta.
Um segundo estudo foi conduzido usando resíduo de comida rico em azoto como subs- trato em processos de escala laboratorial e piloto e que resultou na produção de um tetra polímero de PHA que continha 3-hidroxioctanoato. Um balanço de massa foi feito de forma a compreender o metabolismo dos produtos de fermentação e um criou-se um modelo de pro- jeção das estruturas latentes (PLS) para prever a produção de PHA.
No terceiro estudo, a amónia foi removida de lamas de esgoto fermentadas e alimentada durante a fase de fome como fonte de azoto numa estratégia de alimentação de carbono e
azoto desacoplada. Este passo adicional resultou num aumento de produtividade em 77%
comparativamente a condições de operação similares sem o passo de remoção de azoto.
O impacto do uso de um resíduo de comida rico em azoto como fonte de azoto na fase de fome, enquanto um resíduo de fruta fermentada foi usado como fonte de carbono na fase de fartura foi estudado no quarto estudo. Apesar do pequeno decréscimo no conteúdo final de PHA e produtividade, a mudança de condições de operação permitiu o uso de um resíduo fermentado de baixo custo como alternativa à suplementação de nutrientes.
Esta tese demonstra como ultrapassar desafios de processo através de adaptações ao processo tradicional de forma a produzir um produto de valor acrescimento a partir de resí- duos abundantes.
Palavras-chave: polihidroxialcanoatos, culturas mistas microbianas, resíduo fermentado, mo- nómeros de cadeia média, azoto
C ONTENTS
1 INTRODUCTION ... 1
1.1 From oil-based plastics to environmentally friendly alternatives ... 1
1.2 PHA: structure and properties ... 2
1.3 PHA production using microbial cultures ... 3
PHA metabolism ... 4
1.4 PHA production from waste using mixed microbial cultures ... 6
Acidogenic fermentation... 6
Sequencing batch reactor ... 7
Accumulation step ... 8
1.5 Research gaps in the PHA production process using MMC from wastes ... 8
1.6 Motivation and objectives ... 9
1.7 Thesis outline ... 10
References ... 13
2 AN INTEGRATED PROCESS FOR MIXED CULTURE PRODUCTION OF 3-HYDROXYHEXANOATE-RICH POLYHYDROXYALKANOATES FROM FRUIT WASTE ... 21
2.1 Introduction ... 23
2.2 Materials and methods ... 25
Characterization of fruit waste ... 25
Experimental setup ... 26
Analytical procedures ... 29
Calculations ... 30
2.3 Results and discussion ... 31
Performance of the acidogenic fermentation towards caproate production ... 31
Sequencing batch reactor ... 35
Accumulation reactor ... 37
Kinetic assays ... 39
The fate of caproate ... 44
Implications of the study ... 45
2.4 Conclusions ... 46
Acknowledgments ... 47
References ... 47
3 POLYHYDROXYALKANOATES AS AN ALTERNATIVE FOR URBAN FOOD WASTE VALORIZATION ... 55
3.1 Introduction ... 57
3.2 Materials and methods ... 59
Characterization of the food waste ... 59
Experimental setup at lab scale ... 59
Experimental setup at pilot scale ... 61
PHA accumulation ... 62
Calculations ... 63
3.3 Results and discussion ... 65
Feed characterization ... 65
SBR operation ... 66
Accumulation reactor operation ... 70
Polymer characterization ... 73
Carbon flow in PHA metabolism ... 73
Overall yield of the process ... 78
3.4 Conclusions ... 79
Acknowledgments ... 80
References ... 80
4 IMPROVEMENT IN PHA PRODUCTION FROM FERMENTED SEWAGE SLUDGE THROUGH NITROGEN REMOVAL OPTIMIZATION ... 85
4.1 Introduction ... 87
4.2 Materials and methods ... 89
Characterization of fermented waste sewage sludge ... 89
Experimental setup ... 89
Analytical procedures ... 92
Calculations ... 92
4.3 Results and discussion ... 93
Nitrogen removal experiments ... 93
PHA production 3-stage system operation ... 97
Implications of this study ... 101
4.4 Conclusions ... 102
Acknowledgments ... 102
References ... 103
5 IMPACT OF PARTIALLY UNCOUPLED CARBON AND NITROGEN FEEDING STRATEGY WITH A DUAL FEEDSTOCK ON THE PHA-ACCUMULATING CULTURE USING MIXED CULTURES ... 107
5.1 Introduction ... 109
5.2 Materials and methods ... 111
Characterization of fermented wastes ... 111
Experimental setup ... 111
Analytical procedures ... 113
Calculations ... 114
5.3 Results and discussion ... 114
Response of the selection reactor to the replacement of synthetic nutrients by a N&P rich waste ... 114
Process performance under Conditions 1 and 3 ... 116
Impact of feeding a micronutrients solution (Condition 3 vs Condition 5) ... 119
Implications of the study ... 121
5.4 Conclusions ... 122
Acknowledgements ... 123
References ... 123
6 GENERAL CONCLUSIONS AND FUTURE PERSPECTIVES ... 127
6.1 General conclusions and follow-up work ... 127
6.2 The future of the PHA production process using MMC ... 129
L IST OF F IGURES
Figure 1.1. Generic chemical structure of PHA, where X is the number of carbon atoms in the structure of the monomer and R is the side chain. Adapted from [19]. ... 2 Figure 1.2. Main metabolic pathways involved in PHA storage. PHA can be stored through Entner-Doudoroff pathway (a), de novo fatty acid synthesis (b), β-oxidation of fatty acids (c, d) and alkanes oxidation (e). Adapted from Reis et al. [29]. ... 5 Figure 2.1. Trends of the OLR applied to the reactor and of the concentrations of each FP in the outlet of the acidogenic reactor throughout the study. ... 32 Figure 2.2. Trends of fraction of each gas identified in the headspace of the UASB reactor during operation. ... 33 Figure 2.3. Concentration of FP (a) and PHA content and monomer content in the biomass (b) in a typical accumulation assay. ... 38 Figure 2.4. Trends of FP concentration over time in the kinetic batches at lab scale... 41 Figure 2.5. PHA content in the biomass during the kinetic batch tests at lab scale. ... 42 Figure 2.6. Proposed biosynthesis pathway of P(3HB-co-3HV-co-3HHx) in MMC from short chain fatty acids (solid line) and possible pathway for caproate metabolism in MMC (dashed line). Dotted lines represent reactions from β-oxidation that result in cleavage of the substrate and lead to Krebs cycle. Adapted from Reis et al. and Sudesh et al. [25,53]. ... 46 Figure 3.1. Trend of the concentration of the FP fed in the SBR in the pilot-scale test (Ace, acetate; EtOH, ethanol; Prop, propionate; Lac, lactate; But, butyrate; iBut, iso-butyrate; Suc, succinate; Val, valerate; Cap, caproate; Hep, heptanoate; Oct, octanoate). ... 67 Figure 3.2. FF ratio in SBR operation in lab-scale (A) and pilot-scale (B). ... 68 Figure 3.3. Fitting plots of estimated results of the model vs experimental values for HB, HV, HHx and HO, respectively (in mmol L-1). ... 75
Figure 3.4. Fitting plots of modelled vs experimental values for HB, HV, HHx and HO, respectively (in mmol L-1). ... 77 Figure 3.5. Fitting plot of modelled total PHA vs experimental total PHA calculated through the sum of the model results (in mmol L-1). ... 78 Figure 3.6. Mass flow diagram of PHA production from FFW. ... 79 Figure 4.1. Contour plot by response surface methodology for the Mg-enhanced chemical precipitation of ammonium tests. ... 94 Figure 4.2. Contour plot by response surface methodology for the stripping of ammonia tests:
No aeration (A); Aeration (B). ... 96 Figure 4.3. Nitrogen removal and recovery test: (A) stripping of ammonia and (B) N recovery from the stripped gas stream. ... 97 Figure 4.4. FF ratio during the selection step in both phases of operation. ... 98 Figure 5.1. FF ratio for the different operating conditions in the SBR. ... 115 Figure A.1. Flow chart of the typical 3-stage process. A feedstock rich in organic compounds (e.g. sugars, proteins and/or lipids) is fermented into an effluent rich in fermentation products, which is fed to the subsequent steps. In the second-step a PHA-accumulating culture is selected from waste activated sludge, which is then used in the third step to accumulate PHA.
... 131 Figure A.2. Comparison between a sample of the SBR (red) and a standard (blue). The black box highlights the monomer HHx for both the sample and the standard... 132
L IST OF T ABLES
Table 1.1. R-group of the most common monomers of PHA. These monomers are equivalent to the ones represented in Figure 2.1 with X = 1. ... 3 Table 1.2. Properties of polypropylene and PHA with different monomeric compositions ... 4 Table 2.1. Characterization of the fruit waste in fermentation reactor ... 27 Table 2.2. Composition of the feedstock used in each batch experiment (COD basis). Biomass A corresponds to a caproate-acclimated biomass whereas Biomass B corresponds to a biomass non-acclimated with caproate ... 29 Table 2.3. Microbial community dynamics of 10 most abundant genera of the acidogenic reactor. Values indicate percentage of total number of reads. OUTs listed indicate phylum and the lowest assigned taxonomic classification that could be obtained. G samples correspond to granular biomass, S samples correspond to suspended biomass (p - phylum; g - genus). ... 34 Table 2.4. SBR performance parameters after pseudo-steady state ... 36 Table 2.5. Semi-quantitative FISH characterization of the MMC enriched in the SBR during pseudo-steady state (c – class; g – genus). ... 37 Table 2.6. Summary of the parameters obtained in the accumulation reactor at pilot scale; Feed concentration (gCODFP L-1): [Lac] = 0.09, [Ace] = 1.03, [But] = 1.61, [Val] = 0.03, [Cap] = 11.539 Table 2.7. Summary of the parameters obtained in the kinetic batches at lab scale ... 40 Table 3.1. Preliminary characterization of the hydrolysate obtained from Valorsul ... 60 Table 3.2. Summary of the SBR operating conditions ... 62 Table 3.3. Reactions considered in the carbon balance of the FP... 64 Table 3.4. Summary of the characterization of the FFW that was fed to the reactors ... 66 Table 3.5. Summary of SBR performance parameters after pseudo-steady state ... 71 Table 3.6. Summary of accumulation performance tests carried out after achieving a pseudo- steady state of each operating condition. ... 72
Table 3.7. Characterization of the polymer produced at lab scale (Mw, average molecular weight; PDI, polydispersity index; Tg, glass transition temperature; Tm, melting temperature; Xc, crystallinity fraction) ... 74 Table 4.1. Initial characterization of the FSS used in this study. ... 90 Table 4.2. SBR performance parameters after pseudo-steady state in both phases of operation ... 99 Table 4.3. Summary of the parameters obtained in the accumulation reactor in the two studied phases of operation ... 101 Table 5.1. Characterization of the fermented wastes ... 112 Table 5.2. Summary of the operating conditions in the SBR ... 113 Table 5.3. Summary of the SBR performance ... 117 Table 5.4. Semi-quantitative FISH characterization of the MMC enriched in the SBR during pseudo-steady state ... 118 Table 5.5. Summary of the fed-batch accumulation tests carried out after SBR was stabilized in each operation condition ... 119 Table B.1. Description of the conditions of the experiments conducted for the Mg-enhanced chemical precipitation, where each experiment was performed in duplicate. t (h) corresponds to the duration of the experiment; Mg corresponds to the added Mg2+ to NH4+ molar ratio ... 133 Table B.2. Description of the conditions of the experiments conducted for the stripping of nitrogen, where each experiment was performed in duplicate. t corresponds to the duration of the experiment ... 134 Table B.3. ANOVA results for response surface of the model for the Mg-enhanced chemical precipitation of ammonia tests ... 135 Table B.4. ANOVA results for response surface of the model for the ammonia stripping tests ... 135
G LOSSARY , ACRONYMS AND SYMBOLS
μmax maximum specific growth rate
A.C. After Christ
Ace acetate
Acetyl-CoA acetyl coenzyme A
ADF aerobic dynamic feeding
ANOVA analysis of variance
B.C. Before Christ
But butyrate
C carbon
Cap caproate
CHSOL soluble carbohydrates
CHTOT total carbohydrates
COD chemical oxygen demand
CODSOL soluble chemical oxygen demand CODTOT total chemical oxygen demand CSTR continuous stirred tank reactor
DO dissolved oxygen
DoE design of experiments
EtOH ethanol
FA fatty acids
FF feast to famine
FFW Fermented food waste
FISH fluorescence in situ hybridization FITC fluorescein isothiocyanate
FP fermentation products
FSS fermented sewage sludge
FW fruit waste
GC gas chromatography
GHG greenhouse gas
HA hydroxyalkanoate
HB 3-hydroxybutyrate
Hep heptanoate
HHx 3-hydroxyhexanoate
HO 3-hydroxyoctanoate
HPLC high performance liquid chromatography
HRT hydraulic retention time
HV 3-hydroxyvalerate
iBut iso-butyrate
IC inorganic carbon
iVal iso-valerate
Km affinity constant
Lac lactate
lcl-PHA large-chain length polyhydroxyalkanoate
LV latent variables
MCFA medium chain fatty acids
mcl-PHA medium-chain length polyhydroxyalkanoate
MMC mixed microbial cultures
MW average molecular weight
N nitrogen
NADH reduced nicotinamide adenine dinucleotide
Oct octanoate
OFMSW organic fraction of municipal solid waste
OLR organic load rate
OUT operational taxonomic unit
P phosphorus
PC principal component
PCA principal component analysis
PDI polydispersity index
PHA polyhydroxyalkanoate
PHB polyhydroxybutyrate
PHBHx poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) PHBV poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PLS projection to latent structures
PP polypropylene
PPHA polyhydroxyalkanoate productivity PHHx 3-hydroxyhexanoate productivity
Pro proprionate
ProtSOL soluble protein ProtTOT total protein
qFPMAX specific substrate uptake rate qNMAX specific nitrogen uptake rate qPHAMAX specific PHA storage rate
qXA specific biomass production rate
R2 coefficient of determination
rAce volumetric acetate uptake rate
rAceMAX maximum volumetric acetate uptake rate rBut volumetric butyrate uptake rate
rButMAX maximum volumetric butyrate uptake rate rCap volumetric caproate uptake rate
rCapMAX maximum volumetric caproate uptake rate
rFP volumetric FP uptake rate
rFPMAX maximum volumetric FP uptake rate
rHB volumetric 3-hydroxybutyrate production rate
rHBMAX maximum volumetric 3-hydroxybutyrate production rate rHHx volumetric 3-hydroxyhexanoate production rate
rHHxMAX maximum volumetric 3-hydroxyhexanoate production rate rHV volumetric 3-hydroxyvalerate production rate
rHVMAX maximum volumetric 3-hydroxyvalerate production rate rN volumetric nitrogen uptake rate
rNMAX maximum volumetric nitrogen uptake rate
rNFAMINE volumetric nitrogen uptake rate in the famine phase rNFEAST volumetric nitrogen uptake rate in the feast phase
RNA ribonucleic acid
RMSE root mean square error
RMSECV root mean square error of cross-validation
rPHA volumetric polyhydroxyalkanoate production rate
rPHAMAX maximum volumetric polyhydroxyalkanoate production rate rPro volumetric propionate uptake rate
rRNA ribosomal ribonucleic acid rVal volumetric valerate uptake rate
SBR sequential batch reactor
SCFA short chain fatty acids scl-PHA short-chain length PHA
SRT sludge retention time
Suc succinate
TC total carbon
Tg glass temperature
Tm melting temperature
TKN total Kjeldahl nitrogen
TN total nitrogen
TNSOL soluble fraction of total nitrogen
TOC total organic carbon
TRL technology readiness level
TS total solids
TSS total suspended solids
UASB upflow anaerobic sludge blanket
Val valerate
VFA volatile fatty acids
VS volatile solids
VSS volatile suspended solids
WAS waste activated sludge
WWTP wastewater treatment plant
XA active biomass
Xc crystallinity fraction
YFP/COD acidification yield
YHB/(Ace+But) Conversion yield of butyrate and acetate into 3-hydroxybutyrate YHHx/Cap Conversion yield of caproate into 3-hydroxyhexanoate
YPHA/FP storage yield
YPHA/FPGLOBAL global storage yield
YXA/FPFEAST growth yield on FP in the feast phase YXA/PHAFAMINE growth yield on PHA in the famine phase
1
I NTRODUCTION
1.1 From oil-based plastics to environmentally friendly alterna- tives
Mankind has taken advantage of plastics since 1600 B.C., when ancient Mesoamerican peoples were processing latex produced by Castilla elastica tree [1]. However, it was not until the 19th century A.C. when Alexander Parkes introduced the world's first-ever man-made plastic [2]. Since then, other synthetic polymers have been discovered and in ~1950 mass production of plastics has started and grown substantially, thus making this material one of the most used nowadays [3]. Plastic industry growth has been sustained on the economic growth worldwide, as these materials were used widely in the development of microelectronic, food and water safety and transportation sector, contributing extensively to healthcare and safety of society [4].
Although these materials possess interesting features such as low density, high resistance and well-optimized manufacturing processes, making them some of the most used materials worldwide, their life cycle is a major concern nowadays. These materials are highly resistant to biodegradation and the mode of disposal is currently not the most environmentally friendly:
they are often disposed of through landfilling, which was the fate of a whopping 79% of all plastics ever produced while only 9% were ever recycled and the remainder incinerated [5–7].
As a consequence of the poor discarding of plastics, approximately 10 million tonnes of plastics end up in the oceans annually, of which about 75% come from land-based sources. This mis- managed waste is either disposed in dumps or in open and uncontrolled landfills and it enters the ocean via inland waterways, wastewater outflows or transport by wind and tides, which can lead eventually to major changes in the marine ecosystem [8–10]. On top of the poor misman- agement of these materials, their production rely on finite stocks, such as oil and gas, which
make up more than 90% of this feedstock [11]. In 2019, the plastic industry consumed 6% of the global oil supply and this value is expected to increase up to 20% by 2050, while accounting for 15% of the total global carbon budget [12,13].
Bioplastics have been proposed as environmentally friendly alternatives for the oil-based plastics. These materials can be used in similar applications as traditional plastics and they contain organic carbon of renewable origin (biobased) and/or can be degraded by naturally- occurring microorganisms (biodegradable) [14]. In the case of biobased and biodegradable plastics, they solve the two major problematics of traditional plastics: they are produced from a non-finite renewable source and the impact of the material at the end-of-life is minimized by their biodegradation in the environment. Hence, these polymers represent a way of reduc- ing the carbon footprint of oil-based plastics and they allow for additional waste management options such as composting.
1.2 PHA: structure and properties
Polyhydroxyalkanoates (PHA) are a family of biocompatible polyesters that can be pro- duced by bacteria as energy and carbon storage and be degraded into CO2 and H2O [15]. Its generic chemical structure is represented in Figure 1.1. Since the discovery and description of PHA for the first time by Maurice Lemoigne [16], more than 150 R-hydroxyalkanoid acid mon- omers have been identified and described [17]. These monomers are often categorized accord- ing to their length: short-chain length PHA (scl-PHA) which contain 3-5 carbons; medium-chain length PHA (mcl-PHA) which contain 6-14 carbons; large-chain length PHA (lcl-PHA) which contain 15 or more carbons [18]. Although there are dozens of monomers in each category, the lcl-PHA is the least common while the most studied monomers are either scl-PHA or mcl- PHA (Table 1.1).
Figure 1.1. Generic chemical structure of PHA, where X is the number of carbon atoms in the structure of the mon- omer and R is the side chain. Adapted from [19].
Owing to the structural variability of these polymers, their mechanical and thermal prop- erties vary widely as well. Poly(3-hydroxybutyrate), the most studied PHA polymer, is compa- rable to polypropylene in a few properties, such as the melting temperature and tensile
strength [20,21] (Table 1.2). However, its extension to break is comparatively very low. As a result, copolymers including HB and other monomers such as HV or HHx have arisen as an alternative because they confer the material with different properties, (e.g. a higher extension to break or elastomeric properties), depending on their monomeric composition. Despite of the similarities shared between polypropylene and PHA, biodegradability is exclusive to PHA.
Many microorganisms can externally degrade the polymer into fragments using PHA depoly- merases, which can be taken up and be completely degraded internally [22]. Owing to the variability of the monomeric composition of this polymer, it can potentially replace some of oil-based plastics, specifically for packaging, coating, consumer goods, durable goods and bags [23].
Table 1.1. R-group of the most common monomers of PHA. These monomers are equivalent to the ones repre- sented in Figure 2.1 with X = 1.
R-Group Category Monomer’s name Abbreviature
CH3 scl 3-hydroxybutyrate HB
C2H5 scl 3-hydroxyvalerate HV
C3H7 mcl 3-hydroxyhexanoate HHx
C4H9 mcl 3-hydroxyoctanoate HO
1.3 PHA production using microbial cultures
After the discovery and characterization of PHA, researchers have studied how it could be produced from microbial cultures. Currently, PHA is produced using single culture biopro- cesses, either with wild-type (e.g. C. necator and P. oleovorans) or genetically modified organ- isms, resulting in very high volumetric productivities (up to 5 gPHB L-1 h-1), very high cell densities up to 200 g L-1 as well as PHA contents up to 80-90% of cell dry weight[25]. Depending on the production scale and despite the high yield and productivity of the process, typically the costs of production and downstream processing can be as high as 1.01-5.00 € kgPHB-1, which is still 3-4 times higher than PP and PE, and it has narrowed the use of PHA polymers to high-end applications, where this cost of production is not an impediment [25,26]. The costs of produc- tion are usually attributed to: (1) the substrate, in which refined sugars such as glucose and sucrose are used; oxygen demand, and the need of feeding pure oxygen or the use of pressur- ized tanks to prevent oxygen limitation due to the high biomass concentrations achieved; (2)
downstream processing, where a series of treatments, separation processes and solvents are used to achieve a high degree of purification [27].
Table 1.2. Properties of polypropylene and PHA with different monomeric compositions
Parameter PP PHB PHBV PHBV PHBHx
Refs. [20] [21] [21] [21] [24]
HB content (%, wt.) n.a. 100 97 75 68
Melting temperature (ºC) 176 177 170 137 88
Glass transition temperature (ºC) -10 2 8 -6 -1
Crystallinity (%) 50-70 60 - - -
Tensile Strength (MPa) 38 43 38 30 8
Young’s modulus (MPa) - - 2.9 0.7 101
Extension to break (%) 400 5 - - 856
PHA metabolism
The mechanisms of PHA storage have been extensively studied in the most efficient PHA- accumulating organisms. They use the Entner-Doudoroff pathway to convert glucose into py- ruvate, which is further metabolized to acetyl-CoA. Under unrestricted growth conditions, ac- etyl-CoA enters the Krebs cycle to be oxidized into CO2 and energy, which is used for growth.
However, when the cells undergo nutrient limitation conditions (e.g. lack of N or P), metabolite concentrations become unbalanced and the cell metabolism shifts considerably. Intracellular NADH concentration increases, protein synthesis slows down, the concentration of RNA in the cytoplasm decreases and cells grow slower or not at all [28]. NADH is also an inhibitor of the Krebs cycle, which results in an increase of acetyl-CoA [29]. Because of the lack of a crucial nutrient, the excess carbon is metabolized into acetyl-CoA, which is then redirected for PHA storage (Figure 1.2a).
Other carbon sources (e.g. short chain fatty acids (SCFA) and medium chain fatty acids (MCFA) can also be used for PHA production by bacteria (Figure 1.2c and Figure 1.2d). Alike the metabolism of sugars, these substrates are converted into acetyl-CoA, which enters the Krebs cycle when all nutrients are present. Under restricted growth conditions, bacteria re- direct the excess acetyl-CoA into PHA accumulation. In these conditions, other acyl-CoA com- pounds maybe formed (e.g. valeryl-CoA from valerate or capryl-CoA from caproate) and di- rectly used as precursors, which may result in different in polymers containing different mon- omers.
Most microorganisms cannot carry out more than one of these pathways, and in some cases, a polymer consisting solely of 3-hydroxybutyrate is produced. However, in the case of some microorganisms, co-polymers are produced owing to the variability of the substrates or the PHA synthases they possess. So far, four classes of PHA synthases have been described [30]. Although class I and class II PHA synthases possess only one subunit, they differ in their monomer specificity. Class I uses preferably 3-hydroxyacids with 3-5 carbon atoms, while class II PHA synthase prefer medium chain (6-14 carbon atoms) 3-hydroxyacids. The remaining clas- ses of PHA synthase possess two subunits, one of which they have in common (PhaC) and have a substrate specificity towards short 3-hydroxyacids.
Figure 1.2. Main metabolic pathways involved in PHA storage. PHA can be stored through Entner-Dou- doroff pathway (a), de novo fatty acid synthesis (b), β-oxidation of fatty acids (c, d) and alkanes oxidation
(e). Adapted from Reis et al. [29].
1.4 PHA production from waste using mixed microbial cultures
Current industrial PHA production processes can hardly compete with conventional pe- troleum-based plastics. In high-end applications such as in biomedicine sector, the high cost of production is sometimes offset by a high selling price because of the in-creased biocom- patibility of these PHA-based materials. In the case of cheaper and/or disposable materials, PHA has largely failed to enter the plastics market yet. Hence, in the last 2 decades, mixed microbial cultures and cheaper carbon sources have received more attention as a potentially cost-efficient solution for some of the cheaper oil-based alternatives. The use of mixed micro- bial cultures (MMC) offers the possibility of skipping all sterilization steps and a decrease in aeration costs, while wastes can replace refined sugars as a low-cost carbon source (such as wastes or industrial by products). PHA production comprising MMC typically involve 3 main stages: (1) an acidogenic fermentation of the organic fraction of the waste to produce a mixture of fermentation products (FP), which are the precursors for PHA production; (2) PHA-accumu- lating culture selection phase, where a MMC undergoes a selective pressure to promote favor- able conditions for PHA-producers and allow them to thrive; (3) a PHA accumulation phase where the culture selected in the previous phase is subjected to excess FP in order to accumu- late PHA up to its maximum capacity.
Acidogenic fermentation
Over the last decades, waste streams have been treated using anaerobic digestion, which allows the conversion of the organic fraction of the stream to be converted into CO2 and, the energy-rich compound, CH4. In wastewater treatment plants, this process is carried out by a consortium of anaerobic microorganisms through a series of biochemical processes: (1) en- zyme-mediated hydrolysis of macromolecules such as lipids, proteins and carbohydrates into simpler monomers such as long-chain fatty acids, amino acids and monosaccharides by hy- drolytic bacteria; (2) acidogenic fermentation of the simple organic compounds into fermen- tation products (FP), comprising fatty acids (e.g. acetate, butyrate, caproate), organic acids (e.g.
lactate and succinate), alcohols (e.g. ethanol and 2,3-butanediol) and H2/CO2; (3) conversion of FP into acetate and H2/CO2 by acetogenic bacteria; (4) methanogenic bacteria convert acetate and H2/CO2 into CH4. In the case of the acidogenic fermentation process, in order to produce a FP-rich for PHA production, step 3 and/or step 4 of anaerobic digestion are typically inhibited either by applying a low solids retention time, low temperature, low pH or a combination of these [29].
The manipulation of the operating conditions in the acidogenic fermentation of-ten re- sults in variations of the FP profile, as the microbial culture changes or metabolic pathways within a single microorganism are preferred over others [31]. For instance, a Bengtsson et al.
concluded that an increase in the pH (from 3.5 to 6) in the fermentation of paper mill effluent resulted in an increase in butyrate and propionate production [32]; Contreras-Dávila et al. con- cluded that lactate was the key electron donor for its chain elongation into butyrate and pH >
4 was essential for its production [33]. Since odd-chain FP (e.g. propionate and valerate) are typically known as HV precursors and even-chain FP (e.g. acetate and butyrate) are considered HB precursors, the operating conditions of the acidogenic fermentation must then be tightly regulated, as the fermented effluent produced in this step is used as substrate in the subse- quent two steps and it can have a significant impact on the monomeric composition of the PHA and its characteristics [34].
Sequencing batch reactor
A PHA-accumulating culture is usually enriched from activated sludge from a wastewater treatment plant. Although other reactor configurations have been proposed, this step is typi- cally achieved in a sequencing batch reactor (SBR) [26,35]. A broad consortium of microorgan- isms from activated sludge undergoes cycles of carbon source abundance followed by carbon source depletion. The microorganisms that can produce PHA during the phase of carbon source excess (feast phase) have a competitive advantage over non-PHA producers during the carbon source depletion phase (famine phase), as PHA-accumulating microorganisms are able to grow using the stored PHA and the remaining nutrients during this phase [28]. More spe- cifically, during the famine phase, PHA-accumulating microorganisms grow using the nutrients available and the stored PHA. After nutrient depletion, other intracellular components (e.g.
mRNA, enzymes and NADH) decrease to low levels, decreasing metabolic activity to a mini- mum [29,36]. When a new cycle of external carbon excess (feast phase) begins, cells replenish their NADH levels, triggering the downregulation of the Krebs cycle and driving the substrate preferentially towards PHA storage [36]. Microorganisms that cannot produce PHA will be washed out of the reactor after a few cycles. In order to increase the selective pressure towards PHA-accumulating microorganisms, it was proposed that carbon and nitrogen would be fed separately in the feast phase and famine phase, respectively [37,38]. This approach forces mi- croorganisms to only produce PHA during the feast phase and only grow during the famine phase with the PHA that was stored before because nitrogen is a crucial nutrient for growth and is not available during the phase where exogenous car-bon sources are available.
Accumulation step
The final step of the PHA production process is the production of PHA, which can be accomplished by feeding an excess of substrate obtained in the acidogenic fermentation (first step) to the PHA-accumulating consortium selected in step 2. This step is essentially an ex- tended feast phase, preferably carried out by feeding a nitrogen-depleted substrate to prevent a growth response, hence driving the carbon towards PHA production [39]. The feeding of the substrate is usually carried out in pulse-wise manner and con-trolled by dissolved oxygen (DO) concentration to prevent excess feeding and substrate inhibition.
1.5 Research gaps in the PHA production process using MMC from wastes
PHA production from wastes using mixed microbial cultures is a process currently under development. Over 2 decades of work by several researchers have culminated in hundreds of publications and patents being published and reviewed, disclosing important knowledge and helping to bring the process up to a technology readiness level (TLR) 6-7 [40–42]. Although most of these studies have been carried out at lab scale and many of them still use synthetic carbon sources, there has been an increase in research using wastes and work carried out at pilot-scale, narrowing the gap to full-scale production. Furthermore, a demonstration plant
"PHA2USE" recently opened with the aim at producing Poly(3-hydroxybutyrate-co-3-hy- droxyvalerate) (PHBV) from wastes, which showcases the recent successes in the development of this technology and represents a major step forward towards full-scale implementation of the technology [43].
The work carried out at pilot scale has been either a validation of the successes obtained at lab scale or innovative research. Plenty of the issues, which have so far prevent-ed to scale the technology further up, have been considered in recent research. Valentino et al. studied the impact of treating a combined waste comprising the organic fraction of municipal solid waste and sewage sludge, the effect of temperature on the SBR and the insertion of the PHA- production process into a wastewater treatment plant in the scope of a biorefinery [44–47].
Tamis et al. compared the process at pilot-scale with similar conditions at lab-scale and char- acterize the process at high pH and ammonia concentrations [48–50]. Using fruit waste, Matos et al. has studied the impact of sludge retention time (SRT) and organic loading rate (OLR) in a pilot study and has combined multiple known process conditions to improve the productivity
of the process as a whole, while Sousa et al. developed a technology to measure PHA content online [51–53]. Other authors have studied other feedstocks, modes of operation, the fluctua- tions of the microbial culture [54–58]. Despite the efforts to solve the various challenges of the PHA production process using MMC, the process is still not economically viable as some of challenges still persist, limiting process efficiency and increasing costs of production.
The diversity and heterogeneity of the wastes can bring many challenges, and for that reason, it has been one of the most studied variables of this field in the last decade. Regarding the carbon fraction of the waste, heterogeneity of the feedstock is overcome in the acidogenic reactor by fermenting the waste into a FP-rich fermented feedstock that then is used to pro- duce a PHA containing up to 50% of HV, with the remaining being HB. Polymers containing different HV contents in the polymer have different properties among themselves. However, the fact that MMC often produce only these two monomers limits the properties of the mate- rials produced, the applications where they can be used and possibly the market value. Re- garding the nitrogen fraction of the waste, more often than not, the issue is not addressed at all, as researchers simply use the waste as is without any pre-treatment or process adaptation, resulting in either the simultaneous addition of nitrogen and carbon simultaneously, or the addition of a commercial nitrogen salt.
1.6 Motivation and objectives
PHA are a family of bioplastics that can be completely biodegraded into CO2 and H2O [59]. They possess a wide range of thermal and mechanical properties, which makes them one of the most promising materials to replace the plastics produced from non-renewable sources [25]. Although its market share is expected to increase in the next years [60], PHA is still pro- duced through an expensive bioprocess, which requires chemically defined substrates, sterili- zation and extensive aeration, hindering the commercialization of this material in low-end ap- plications [51].
A low-cost process using MMC as a PHA-accumulating culture, which has been un-der development for at 25 years now [61], allows the production of PHA at a reduced cost, albeit with a lower productivity than single culture systems [29]. In this bioprocess, sterilization is not required, and the volumetric aeration requirements are lower when com-pared to pure culture PHA processes. The use of wastes has been considered as an alternative to chemically-defined media (e.g. glucose and acetate) because of the potential cost reduction associated (40-50%) [62]. However, owing to the complexity of these feedstocks, negative effects on PHA
production process may occur, decreasing production rates or increasing instability on the process. Nitrogen-rich substrates can have a negative effect on PHA production process [7], while non-fermented and/or slowly biodegradable organic matter could possibly promote the growth of non-PHA accumulators [8]. The complexity of the feedstocks also results in a PHA- rich biomass containing a wider variety of impurities, contributing to harder and more expen- sive extraction process. Unless these issues are solved by pre-treating or co-valorizing the waste further somehow, the eco-nomic viability of producing this material to be used in low- cost applications will hardly ever be achieved.
The objective of this thesis is to test 3 different feedstocks and develop strategies to explore their unique characteristics in order to either improve process performance or produce novel materials. This knowledge could be used as a stepping point for further research and eventually full-scale implemented in the industry sector. The specific goals of this thesis are the following:
1. Produce added-value 3-hydroxyhexanoate-containing PHA using fruit waste as feedstock.
2. Use a nitrogen-rich food waste as feedstock to produce PHA, without feed-stock pre-treatment, at laboratory and pilot-scale.
3. Pre-treat a nitrogen-rich waste activated sludge to be used as feedstock for PHA production.
4. Test two fermented feedstocks simultaneously as carbon (nitrogen-free) and nitro- gen source in the feast-famine strategy.
1.7 Thesis outline
This thesis is divided into six chapters, as follows:
Chapter 1: An introduction to background and state-of-the-art of the PHA production technology and its current research needs, including the definition of motivation, objectives and thesis’ outline
Chapter 2: Depending on the fermentation conditions, a waste can be converted into an effluent enriched in a specific FP, which can lead to different FP profiles. In this study, the aci- dogenic conditions were tailored towards caproate production, so that it could be used as precursor for HHx-enriched PHA. The microbial consortium that led to the caproate-rich efflu- ent was identified. Several fed-batch tests were carried out with aim of understanding the con- version of caproate into HHx and its biochemical mechanism was elucidated.
This work was published in an international peer-reviewed journal as: Fernando Silva, Mariana Matos, Bruno Pereira, Cláudia Ralo, Daniela Pequito, Nuno Marques, Gilda Carvalho, Maria. A. M. Reis, 2021, An integrated process for mixed culture production of 3-hydroxyhex- anoate-rich polyhydroxyalkanoates from fruit waste, Chemical Engineering Journal, 427, 1319908. https://doi.org/10.1016/j.cej.2021.131908
A provisional patent was submitted as: Polyhydroxyalkanoates and methods thereof (Provisional Patent Application Nº 116619 at National Institute for Industrial Property rights submitted on July 31, 2020)
Chapter 3: Complex feedstocks can often be a limitation in the PHA production process as they are often enriched in inhibiting compounds such as ammonia, non-readily biodegrada- ble organic matter, solids or ethanol. The use of nitrogen-rich wastes in the SBR typically results in a more balanced storage and growth responses, which can be detrimental to the process. In this study, a nitrogen-rich fermented food waste was used as feedstock to study the PHA pro- duction process. Initially, the OLR was studied at bench scale, before moving to pilot scale where the impact of HRT on the reactor was evaluated. The metabolism of the FP into PHA was studied using carbon mass balances and a model.
This work will be submitted to an international journal as: Fernando Silva, Gianluca Gili- berti, Mariana Matos, Cláudia Galinha, Christian Grandfils, Madalena Dionisio, Gilda Carvalho, Maria. A. M. Reis, Polyhydroxyalkanoates as an alternative for urban food waste valorization.
Chapter 4: Following on the work of Chapter 3, approaches to remove nitrogen from the feedstock were considered to improve the selection of PHA-accumulating culture. In this study, Mg-enhanced precipitation of ammonia and ammonia stripping were considered as pre-treat- ments for a fermented waste activated sludge (WAS). A SBR was inoculated, fed with non- treated WAS and then with treat WAS and the PHA production performance was evaluated after each phase.
This work will be submitted to an international journal as: Fernando Silva, Giovanni Loi, Mariana Matos, Jorge Santos, Gilda Carvalho, Maria. A. M. Reis, Improvement in PHA produc- tion from fermented sewage sludge through nitrogen removal optimization
Chapter 5: Following on the work of Chapter 2 and Chapter 3, an alternative approach to handle nitrogen-rich fermented wastes was considered. In this study, a SBR was inoculated and fed with a carbon-rich, nitrogen-depleted waste and a nitrogen-rich waste separately, which were the main sources of nutrients for the feast and famine phases, respectively. Because of the high solids content of the nitrogen-rich waste, the impact of the HRT was also studied in this test.
This work will be submitted to an international journal as: Fernando Silva, Filipa Pedro, Mariana Matos, Gilda Carvalho, Maria. A. M. Reis, Impact of partially uncoupled carbon and nitrogen feeding strategy with a dual feedstock on the PHA-accumulating culture using mixed cultures
Chapter 6: General conclusions of this PhD project as well as perspectives for future work in the field of PHA production using MMC
Other relevant publications not included in this thesis:
Federico Battista, Giuseppe Strazzera, Francesco Valentino, Marco Gottardo, Marianna Villano, Mariana Matos, Fernando Silva, Maria A.M. Reis, Joan Mata-Alvarez, Sergi Astals, Joan Dosta, Rhys Jon Jones, Jaime Massanet-Nicolau, Alan Guwy, Paolo Pavan, Da-vid Bolzonella, Mauro Majone - 2022 - New insights in food waste, sewage sludge and green waste anaerobic fermentation for short-chain volatile fatty acids production: A re-view - Journal of Environmen- tal Chemical Engineering - 10, 5, 108319, https://doi.org/10.1016/j.jece.2022.108319
Sousa, Beatriz V., Silva, Fernando, Reis, Maria A.M., Lourenço, Nídia D. - 2021 - Moni- toring pilot-scale polyhydroxyalkanoate production from fruit pulp waste using near-infrared spectroscopy - Biochemical Engineering Journal - 176, 108210, https://doi.org/10.1016/j.bej.2021.108210
Mariana Matos, Rafaela A.P. Cruz, Pedro Cardoso, Fernando Silva, Elisabete B. Freitas, Gilda Carvalho, Maria A.M. Reis – 2021 - Sludge retention time impacts on polyhydroxyalka- noate productivity in uncoupled storage/growth processes - Science of the Total Environment, 799, 149363, https://doi.org/10.1016/j.scitotenv.2021.149363
Mariana Matos, Rafaela A. P. Cruz, Pedro Cardoso, Fernando Silva, Elisabete B. Freitas, Gilda Carvalho, Maria A. M. Reis – 2021 – Combined Strategies to Boost Polyhydroxyalkanoates Production from Fruit Waste in a Three-Stage Pilot Plant - Sustainable Chemistry & Engineer- ing, 9, 24, 8270-8279, https://doi.org/10.1021/acssuschemeng.1c02432
Beatriz Meléndez-Rodríguez, Sergio Torres-Giner, Maria A.M. Reis, Fernando Silva, Mar- iana Matos, Luis Cabedo, José María Lagarón – 2021 - Development and Characterization of Blends of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with Fruit Pulp Biowaste Derived Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) of Application Interest in Organic Recyclable Food Packaging, Polymers, 13, 1155, https://doi.org/
10.3390/polym13071155
Maria Luisa Astolfi, Elisabetta Marconi, Laura Lorini, Francesco Valentino, Fernando Silva, Bruno Sommer Ferreira, Silvia Canepari, Mauro Majone – 2020 - Elemental concentration and
migratability in bioplastics derived from organic waste, Chemosphere, 259, https://doi.org/10.1016/j.chemosphere.2020.127472
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