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De Farias Silva, Carlos Eduardo (2017) Exploitation of microalgal biomass as an alternative source to bioethanol production. [Ph.D. thesis]

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Abstract (english)

The use of natural sources in economic activities can aid in the resource saving and recycling and reuse of wastes, contributing for a more sustainable world by providing clean technologies in the industrial and agricultural sector in both developed and developing countries.
In general, increased and improved global strategies for energy safety, security and mitigation of CO2 emissions from energy production processes are required, especially those aimed at maximizing the energy efficiency by expanding the use of clean energy. This means using fuels that are able to implement the carbon cycle without changing the atmospheric balance (renewable fuels), by developing energy resources in CO2 reduced/neutral systems (Brennan and Owende, 2011; Moraes et al., 2017).
The expansion of biofuels production and use is an important issue since it plays primary role in reducing global the climate change. But, in order to insert a new source/technology in the market, several factors are involved such as industrial aspects and economic feasibility, legal restrictions and incentives, international trade, land use, raw material availability and management techniques.
At present, ethanol is the main biofuel produced worldwide. Between 2007 and 2015 bioethanol throughput practically doubled, reaching 25 billion gallons per year, even though after 2010 the production was stagnant (AFDC, 2016). This is the result of a number of reasons, to cite:
- high dependency on the first-generation crops which need a lot of arable land and compete directly with food/feed production;
- need for a complete validation of the lignocellulosic ethanol industry due to unsuitability of the large-scale process because of corrosion problems (mainly in the pretreatment), cost of enzymes, difficult/inhibition of the fermentation step;
- difficulty to utilize all lignocellulosic fractions, according to a biorefinery approach, because each biomass has its biological complexity and the related lignocellulosic content/arrangement/recalcitrance changes significantly;
- Lacking of investments/incentives (mainly, governmental) after the decrease of petroleum prices occurred at the end of 2014.
In fact, based on the type of biomass, bioethanol production is classified as first (raw material saccharine or starch-based – sugarcane and corn); second (lignocellulosic materials); third (microalgal/macroalgal biomass) and fourth (genetically modified cyanobacteria) generation.
Sugar cane ensures the lowest bioethanol production costs. In spite of its significant advantage, it is not a viable option for all the regions of the planet owing to climatic and soil limitations (Belincanta et al., 2016). Consequently, countries of the northern hemisphere have been incessantly looking for new technological routes that permit the efficient production of biofuels while respecting environmental and economic sustainability issues, and ‘new’ generations of biomass-to-ethanol processes are proposed. In addition, countries as Brazil have their sugarcane cultivation saturated, i.e., there is no new extensions of arable land to expand significantly the Brazilian ethanol industry.
Low production costs are the advantage of first generation bioethanol, with the exception of corn-based one, which has a well-established and economically sustainable technology, while second generation still requires more investigations to become economically competitive, with pretreatment and hydrolysis processes needing to be more effective and largely scalable (Gupta and Verna, 2015). On the other hand, micro and macroalgae have not reached a maturity for designing and operating industrial scale plants yet. Therefore, in the case of third and fourth generation bioethanol, further studies are required to develop a competitive and consolidated technology, taking into account also issues other than technological ones.
In third generation bioethanol, microalgae and/or macroalgae biomass are used, which do not have lignin in their cellular structure, and are cultivated with higher growth rates when compared to higher plants. As for this biomass, a suitable process is not available yet, and the related costs cannot be properly estimated. Researchers are currently trying for microalgae: to optimize microalgal productivity and cultivation conditions, as this represents the highest production costs, considering that hydrolysis and fermentation are instead easier compared with lignocellulosics and macroalgae (Jonh et al., 2011; Wei et al., 2013; Hong et al., 2014).
Thanks to their high growth rate, and relatively simple biochemical composition (partitioned among carbohydrates, lipids and proteins), microalgae are acknowledged as very promising feedstock for bioethanol production (Chen et al., 2013). Main aspects needing to be developed in this respect are: carbohydrate cultivation (productivity), hydrolysis and ethanolic fermentation and nutrient recycling/recovery from residual medium/biomass.
With regard to the open issues recalled above, the aim of this research project has been to address and study how to improve the knowledge and discuss the real potentiality of microalgal biomass as a feedstock for an effective bioethanol production, from a perspective of biomass/carbohydrate productivity (microalgal cultivation) and bioconversion process (hydrolysis and fermentation) in a context of a biorefinery concept. In fact, experimental values about fermentation applications from microalgae are not expanded yet in literature. The topics addressed by this thesis are organized and subdivided in twelve chapters as follows.
In Chapter 1, a literature survey to collect and discuss the available information about bioethanol from photosynthetic microorganisms, and to delimit the main lacks to be developed, is done.
Chapter 2 shows a basic analysis of an ethanol biorefinery scheme aimed to include microalgal biomass, discussing the main bottlenecks and the processes which must be developed to adequately evaluate the potentiality of this type of biomass for industrial fermentation proposes.
Chapter 3 treats specifically of the carbohydrate-rich biomass cultivation from microalgae utilizing nutritional and environmental techniques. Operation mode of microalgae cultivation is discussed as well, and the importance to consider semi-continuous and continuous processes is shown, because batch mode is extensively used but less efficient.
Chapter 4 develops a design procedure of a two-unit system composed by a reactor and settler, discussing the influence of operating variables and their limiting values. Specifically, recycle ratio and purge flow rate concepts and effects are extensively studied.
In Chapter 5, the carbohydrate cultivation with Synechococcus PCC 7002 is optimized with respect to the carbon source and pH, because a stable pH (greatly influenced by the carbon source) is necessary for this strain and organic buffers exhibit toxicity. An inorganic buffer study (CO2-bicarbonate) is developed and detailed.
Chapter 6 shows S. PCC 7002 treating urban wastewater to remove chemical oxygen demand, nitrogen and phosphorous content, thus ensuring a double gain: environmental enhancement and valorization of cyanobacterial biomass.
In Chapter 7, continuous cultivation of Chlorella vulgaris in flat-plate photobioreactors to improve carbohydrate productivity is assessed and evaluated using nitrogen limitation as a combination between nitrogen concentration inlet, light intensity and residence time under constant light intensity.
Chapter 8 demonstrates that a similar approach used for the continuous cultivation of C. vulgaris is applicable also to Scenedesmus obliquus. Additionally, it is proved that under outdoor conditions (seasonal regime of illumination – summer and winter), a high carbohydrate content can be produced as well.
In Chapter 9, the kinetics regarding acidic hydrolysis to biomass solubilization and sugars depolymerization is studied with Chlorella vulgaris biomass. An n order kinetics for biomass solubilization and m order for acid concentration is applied for biomass solubilization, providing values of reaction order and activation energy for microalgae. In addition, a saccharification model based on the Michaelis-Menten model is proposed and validated.
Chapter 10 demonstrates how the kinetics considerations determined in the previous chapter can be efficiently applied with the concept of severity factor – CSF (combination between time, temperature and acid concentration). A literature discussion about some assumptions so far considered and the importance to know the biomass nature to determine a coherent range of CSF is provided.
Chapter 11 reports ultrasonication as an effective pretreatment method to improve enzyme accessibility and promote a high rate of hydrolysis from Scenedesmus obliquus biomass. Pretreatment time, ultrasonication intensity and biomass concentration are specifically studied in order to minimize the energy consumption since the bottleneck of the pretreatment method is a high energy dissipation.
In Chapter 12, ethanolic fermentation is addressed with acidic and enzymatic hydrolysates. A systematic optimization of inoculum concentration and consortium between Saccharomyces cerevisiae and Pichia stipitis is determined. Then, the influence of salinity/matrix characteristics was evaluated to understand possible interferences during fermentation process and exhibited lower biochemical yields than the control conditions. Thus, further fermentations experiments are necessary.

Abstract (italian)

L’obiettivo generale di questo progetto di ricerca è stato di verificare la potenzialità delle microalghe come fonte alternativa di biomassa per la produzione di etanolo. In particolare, sono state discusse teoricamente, sperimentalmente e tramite simulazione di processo la coltivazione, l’idrolisi e la fermentazione della biomassa microalgale.
Inizialmente, grazie ad un’ampia ricerca bibliografica ed a prove preliminari effettuate nel Laboratorio Microalghe del Dipartimento di Ingegneria Industriale della Università di Padova si è dimostrato che le specie più promettenti da studiare erano Synechococcus PCC 7002, Chlorella vulgaris e Scenedesmus obliquus, grazie alle loro elevate velocità di crescita e capacità di accumulo di carboidrati, che costituiscono le materia-prima per la produzione di etanolo (fino al 50-60% del peso secco).
In particolare, l’attenta analisi della letteratura riguardo a queste specie ha consentito di verificare che:
- per la produzione di carboidrati è preferibile sviluppare un processo continuo, perché richiede un solo step, mentre il processo batch ne richiede due, e perciò consente di ottenere produttività significativamente inferiori;
- sono disponibili pochi lavori sulla possibilità di usare le microalghe in un processo continuo di questo tipo, mentre sono parecchi i riferimenti al processo batch;
- mancano informazioni sulla capacità di produrre carboidrati da parte di S. PCC 7002.
In una prima parte del lavoro sono stati quindi pianificati e condotti esperimenti in modalità batch con S. PCC 7002, per studiare come mantenere la stabilità e vitalità della coltura durante tutto il periodo di coltivazione. Si sono rilevati problemi con il controllo del pH, ed é stato approfondito l’uso di bicarbonato come fonte di carbonio assieme ad un tampone inorganico, dimostrando in un primo lavoro che il suo impiego è efficiente per la produzione di biomassa ma insufficiente per accumulare un alto contenuto di carboidrati, a causa di una significativa inibizione osmotica causata dall’alta concentrazione di sodio in soluzione. D’altro canto, l’applicazione di un tampone con sostanze organiche, generalmente usato nella coltivazione di microalghe e cianobatteri, ha evidenziato notevoli fenomeni di tossicità per questa specie. Al contrario, il tampone inorganico CO2-bicarbonato messo a punto successivamente è stato capace di garantire la stabilità del pH durante 12 giorni di coltivazione, ed ha consentito di ottenere 6 g L-1 di biomassa (peso secco) con circa il 60% di contenuto di carboidrati.
La coltivazione in continuo di C. vulgaris in un fotobioreattore piatto e sottile è stata studiata per verificare la produzione di carboidrati secondo questa modalità operativa. Il lavoro ha evidenziato l’importanza della riduzione della concentrazione di azoto in entrata al reattore, che va rapportata ai valori di intensità di luce e tempo di residenza per massimizzare la produzione di carboidrati. Si sono misurati valori massimi per la produttività di biomassa e di carboidrati pari a 0.7 e 0.37 g L-1 giorno-1. La stessa procedura é stata usata nello studio del comportamento di S. obliquus, per vedere se l’approccio era valido anche durante la coltivazione all’aperto, simulando la fornitura della luce in modo stagionale. S. obliquus ha mostrato una produttività quasi tre volte maggiore che Chlorella, raggiungendo valori di 0.8 g L-1 giorno-1 (con luce costante) e di 0.71 g L-1 giorno-1 (nell’estate). Questa produttività di carboidrati, se estrapolata a dimensioni industriali, consentirebbe di ottenere tra 45–100 tonbiomass ha-1 anno-1, ben di più di quanto prodotto con le fonti tradizionali di carboidrati.
Un sistema reattore-sedimentatore con riciclo parziale di biomassa è generalmente usato a livello industriale in processi di coltivazione e/o fermentazione. Questo sistema fornisce semplicità e diversi vantaggi per la produzione su larga scala. É stato quindi messo a punto un modello per la simulazione di tale processo, nel caso specifico delle microalghe, per verificare l’influenza dei gradi di libertà (tempo di residenza, rapporto di riciclo della biomassa, età della biomassa e sua velocità di sedimentazione) sulle prestazioni. I principali risultati sono:
- la definizione di un rapporto di riciclo minimo Rmin, di un intervallo operativo per la stessa variabile, e di un valore massimo per la portata di spurgo di biomassa Fwmax;
- la dimostrazione che la perdita di biomassa dalle sommità del sedimentatore abbassa significativamente le prestazioni del sistema;
- la costruzione di grafici adimensionali che legano R a θc/θ e FI/FW (età della biomassa/tempo di residenza, e rapporto tra le portate di ingresso e di spurgo);
- il confronto fra il modello rigoroso messo a punto ed il modello semplificato generalmente considerato in letteratura.
Synechococcus è stata coltivata in acque reflue urbane (sintetiche e reali, con valori di COD pari a 340.0 ± 14.1 mg L-1, di azoto totale pari a 31.0 ± 1.4 mg L-1, e di fosforo totale a 8.20 ± 0.99 mg L-1), con l’obbiettivo di ottenere la depurazione da questi inquinanti. Questa specie è stata molto efficiente nella rimozione di COD, azoto e fosforo totale, raggiungendo valori sotto i limiti di legge in due giorni di coltivazione. L’acqua reflua sintetica ha evidenziato una limitazione dei micronutrienti quando la concentrazione di COD era elevata, differentemente dell’acqua reflua reale, in cui Synechococcus è cresciuta più velocemente.
Successivamente, l’idrolisi e la fermentazione di biomassa microalgale sono state studiate con riferimento ai processi di saccarificazione acida ed enzimatica, e con riferimento ai microorganismi Saccharomyces cerevisiae e Pichia stipitis, rispettivamente. L’idrolisi acida, con acido solforico 0-5% v/v, è stata condotta a diverse temperature (110-130 °C) e tempi di reazione (0-60 min) partendo da 100 g L-1 di concentrazione di biomassa (Chlorella vulgaris). Gli zuccheri idrolizzati sono stati recuperati con un valore massimo pari al 92%, ottenuto con il 3% di acido e 20 min di reazione a 120 °C. La solubilizzazione di biomassa ha esibito un ordine di reazione n = 3.63 ± 0.18 ed un’energia di attivazione pari a 41.19 ± 0.18 kJ/mol. Questi valori sono significativamente diversi di quelli trovati per l’idrolisi di matrici lignocellulosiche, generalmente considerata di primo ordine con Ea = 100-200 kJ/mol, e dimostrano che la biomassa microalgale è più suscettibile al trattamento termico catalizzato all’acido in confronto ai lignocellulosici. Un’equazione basata sulla cinetica di Michaelis-Menten modificata per tenere conto della concentrazione di acido è riuscita a modellare tutti i risultati sperimentali, con un valore della costante di semi-saturazione per la biomassa PolKM pari al 42% della concentrazione iniziale, e con una resa di fermentazione di circa il 60%.
Prima di realizzare l’idrolisi enzimatica, si é reso necessario procedere ad un’ottimizzazione del pretrattamento della biomassa. È stata studiata l’ultrasonicazione applicando un piano statistico di sperimentazione su tre livelli con 3 esperimenti centrali (in tutto si sono condotte 11 prove). Le variabili ottimizzate sono state l’intensità, il tempo di pretrattamento e la concentrazione di biomassa. I risultati hanno dimostrato che l’intensità e il tempo di trattamento sono più importanti e consentono di ottenere un recupero degli zuccheri superiore al 90%, in 4-8 ore. Si é visto che l’energia spesa nel processo di ultrasonicazione non è direttamente collegata con l’efficienza dell’idrolisi, per cui questa può essere condotta efficientemente anche riducendo il consumo di energia nel pretrattamento.
Infine, si sono eseguiti esperimenti di fermentazione dell’idrolizzato ad etanolo con le due specie menzionate (S. cerevisiae e P. stipitis). Si sono ottimizzati la concentrazione di inoculo (7.5 g L-1) ed il consorzio (25% Pichia + 75% Saccharomyces) per avere una produttività tra 5 e 10 g L-1 ora-1 (prossimo al valore industriale). Si è però visto che le velocità di fermentazione sono però più basse a causa di una inibizione dovuta alla accresciuta salinità dell’idrolizzato, un fattore. Per questo motivo, la parte di fermentazione necessita di essere più approfondita al fine di validare l’impiego di questo tipo di biomassa a livello industriale.

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EPrint type:Ph.D. thesis
Tutor:Bertucco, Alberto
Ph.D. course:Ciclo 30 > Corsi 30 > INGEGNERIA INDUSTRIALE
Data di deposito della tesi:11 January 2018
Anno di Pubblicazione:31 October 2017
Key Words:bioetanolo, coltivazione di microalgale, cinetica, modelli, fermentazione bioethanol, microalgal cultivation, kinetics, model, fermentation
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/13 Biologia applicata
Area 09 - Ingegneria industriale e dell'informazione > ING-IND/27 Chimica industriale e tecnologica
Area 03 - Scienze chimiche > CHIM/11 Chimica e biotecnologia delle fermentazioni
Struttura di riferimento:Dipartimenti > Dipartimento di Ingegneria Industriale
Codice ID:10643
Depositato il:14 Nov 2018 14:16
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