“Obtaining and selecting microalgal genotypes suitable for the bioremediation of matrices containing hydrocarbons, starting from Haematococcus pluvialis (Flotow,1844)”

The world economy, even today, is based on the use of fossil fuels to obtain energy and specifically coal and oil. Oil consumption in 2020 increased by 0.9 million barrels per day while the demand for liquid fuels reached historic highs reaching 100 million barrels per day. The use of oil governs stock exchanges and world markets few and specific cartels determine the selling price while maintaining a sort of economic monopoly. Precisely for this reason, the extraction and refining of crude oil remain an extremely intense activity. Basilicata, a region of southern Italy, in particular, represents the largest onshore site on the European continent, contributing alone to 63% of Italian crude oil production. The process of extraction, refining and transport, however, are crucial points for the environmental pollution of soil, air and water. During this thesis paper, we focused on water pollution, caused both by transport (ships and/or underwater pipelines) subject to numerous accidents, and by extraction, during which processing waters come into contact with toxic hydrocarbons and harmful. Petroleum consists of aliphatic and non-aliphatic hydrocarbons, as well as other elements, main heteroatoms such as sulfur and nitrogen. Furthermore, hydrocarbons can be highly toxic: volatile hydrocarbons (VOCs) such as benzene, toluene, ethylbenzene and xylene (BTEX), or polycyclic aromatic hydrocarbons (PAHs) are the cause of the onset of various neurological, respiratory and tumour pathologies. It is therefore evident that the entire industrial process must be regulated to avoid contact with these compounds and that, if accidental spills occur, they must be immediately confined and treated. The methodologies used for this purpose include physicochemical processes that break down the molecules of the crude oil, making it more sensitive to attacks by atmospheric and natural agents. Among these, the evaporation of low molecular weight molecules, the emulsion of the oily fraction with water, solubilization and sedimentation contribute in part to the removal of the crude oil, but there is also a great contribution from the microbiological world, especially prokaryotic, which through bioremediation processes degrades and removes many toxic compounds. The limitations of these technologies lie in the use of chemical compounds that are more harmful to the ecosystem than the oil itself and the saturation of microorganisms that can act only on certain compounds. Precisely by the need to find a methodology that can constitute an innovative solution to the aforementioned problems, microalgae were thought of. Microalgae are unicellular, microscopic, photosynthetic and photoautotrophic organisms capable of removing carbon dioxide from the atmosphere, using it in the photosynthesis process to produce energy and release oxygen. There are different classes of microalgae that can also be placed in different domains (prokaryotes and eukaryotes), with different characteristics and physiologies, but the object of our study was green, eukaryotic and photoautotrophic microalgae. Their ability to use organic carbon, modifying their metabolism, thus becoming mixotrophic, has made them excellent candidates for their use in the world of bioremediation. Several studies have focused on their use for bioremediation, for example, some microalgae have been able to remove sulphate, and nitrates in significant percentages. The most investigated microalgae in this area was Chlorella spp. which removed after 5 days of treatment, 80% of oil emulsified in the middle in a 2020 study, and efficiently reduced the PAHs present in a 2013 study. In this thesis, the analyzed microalgae was Haematococcus pluvialis, known to the scientific world for its ability to produce one of the most powerful antioxidants in nature, astaxanthin (ASX) but never considered for the bioremediation of hydrocarbons of petroleum origin. This PhD project aimed to research a new and innovative technology that, thanks to the use of microalgae, can effectively remove petroleum hydrocarbons in aqueous matrices, creating different genotypes capable of acting more efficiently than the unchanged genotype. (WT). Thanks to a selective pressure process, H. pluvialis was induced to mutate and adapt to an extremely toxic condition. Once several mutant strains were selected, they were followed in their growth and their ability to produce astaxanthin, always comparing them with WT. Since our knowledge did not allow us to determine whether the mutations that occurred were genetic or epigenetic, a genetic analysis was also carried out through RAPD-PCR on the transcript to highlight the diversity of expression of the different genotypes between them and with the non-mutated species. under conditions of non-stress and in subsequent generations. It was thus possible to obtain a dendrogram through “cluster analysis” which highlighted the genetic expression distances between them.This result indicates that the difference in expression under standard conditions is attributable to underlying genetic modifications. Subsequently, after trying different stress and bioremediation conditions, they were grown in the presence of 1% of pure crude in anaerobic conditions, to evaluate the removal and/or degradation activity. The chemical analyses carried out only after 10 days of treatment immediately showed that the amount of carbon present in the culture medium had almost doubled while other parameters such as fluorides, chlorides and sulphates decreased. After 40 days of treatment, preliminary analyzes by GS-MS and GC-FID showed that both in the aqueous medium and in the algal biomass, there were hydrocarbons and oily substances, results confirmed, then, by a quantitative analysis at 20 and 160 days in which, with different kinetics, a decrease in hydrocarbons is highlighted, which for the mutant strain PA1004 had been removed both from the medium and from the biomass. Furthermore, after 120 days the genetic analysis was repeated to evaluate any adaptive mechanisms that occurred following the stress induced by oil. By maintaining the identical amplification conditions (primers, temperatures and concentrations), the result of the "cluster analysis" showed that, indeed, there had been changes in genetic expression, leading to a reorganization of the distances previously obtained in optimal conditions. These distances reflect the behaviour of the genotypes in the presence of the exogenous agent. Some have completely removed the oil, others have acted only on the oily fraction, helping to create a bituminous particulate by grouping as per the generated dendrogram. The absence of hydrocarbons within the aqueous medium and algal biomass is in itself an extremely interesting result, confirming the objectives set at the beginning of this research path. Not only was H. pluvialis able to survive in the presence of crude oil, but also the genotypes generated, proved to be a promising alternative in hydrocarbon bioremediation. In the future, we prepare ourselves to analyze not only the pathways involved in the mutations, to understand the mechanisms underlying this process, but also to analyze the by-products generated to reuse them in a circular economy perspective suitable for the removal and reuse of a potentially harmful product. for man and the environment.L’economia mondiale, ancora oggi, si basa sull’utilizzo di combustibili fossili per ricavare energia e nello specifico carbone e petrolio. Il consumo di petrolio nel 2020 è aumentato di 0.9 milioni di barili al giorno mentre la domanda di combustibili liquidi ha raggiunto massimi storici arrivando a 100 milioni di barili al giorno. L’utilizzo del petrolio governa borse e mercati mondiali, infatti pochi e specifici cartelli ne determinano il prezzo di vendita mantenendo una sorta di monopolio economico. Proprio per questo, l’estrazione e la raffinazione del greggio rimane ancora un’attività estremamente intensa. La Basilicata, regione del sud Italia, in particolare, rappresenta il sito onshore più grande del continente europeo, contribuendo da solo al 63% della produzione di greggio italiana. Il processo di estrazione, di raffinazione e di trasporto, tuttavia, costituiscono dei punti nevralgici per l’inquinamento ambientale di suolo, aria e acqua. Durante questo elaborato di tesi ci si è soffermati sull’inquinamento delle acque, causato sia dal trasporto (navi e/o condutture sottomarine) soggetto a numerosi incidenti, sia dall’estrazione, durante la quale acque di lavorazione vengono in contatto con idrocarburi tossici e nocivi. Il petrolio è costituito principalmente da idrocarburi alifatici e non, oltre che da altri elementi, principalmente eteroatomi come zolfo e azoto. Gli idrocarburi, inoltre, possono essere altamente tossici: gli idrocarburi volatili (VOCs) come il benzene, il toluene, l’etil-benzene e lo xilene (BTEX), o gli idrocarburi policiclici aromatici (PAHs) rappresentano la causa dell’insorgenza di diverse patologie neurologiche, respiratorie e tumorali. Risulta evidente, quindi, come tutto il processo industriale vada regolamentato per evitare contatti con questi composti e che, qualora avvengano, fuoriuscite accidentali, queste debbano essere immediatamente confinate e trattate. Le metodologie utilizzate a tal scopo comprendono processi fisico-chimici che rompono le molecole del greggio, rendendolo più sensibile agli attacchi degli agenti atmosferici e naturali. Tra queste l’evaporazione delle molecole a basso peso molecolare, l’emulsione della frazione oleosa con l’acqua, la solubilizzazione e la sedimentazione contribuiscono in parte, alla rimozione del greggio, ma vi è un grande contributo anche da parte del mondo microbiologico, soprattutto procariotico, il quale tramite processi di bioremediation degrada e rimuove molti composti tossici. I limiti di queste tecnologie risiedono nell’utilizzo di composti chimici che risultano essere più dannosi del petrolio stesso per l’ecosistema e la saturazione dei microrganismi che riescono ad agire solo su determinati composti. Proprio in virtù della necessità di trovare una metodologia che possa costituire una soluzione innovativa alle problematiche sopracitate, si è pensato alle microalghe. Le microalghe sono organismi unicellulari, microscopici, fotosintetici e fotoautotrofi in grado di rimuovere anidride carbonica dall’atmosfera, utilizzandola nel processo di fotosintesi per produrre energia e rilasciare ossigeno. Esistono diverse classi di microalghe collocabili anche in diversi domini (procarioti ed eucarioti), con caratteristiche e fisiologie diverse, ma l’oggetto del nostro studio sono state le microalghe verdi, eucariotiche e fotoautotrofe. La loro capacità di utilizzare il carbonio organico, modificando il loro metabolismo, divenendo così mixotrofici, le ha rese ottime candidate per il loro impiego nel mondo della bioremediation. Diversi studi si sono concentrati sul loro utilizzo per la bioremediation, ad esempio alcune microalghe sono state in grado di rimuovere solfato, e nitrati in percentuali significative. La microalga più investigata in questo ambito è stata Cholrella spp. che ha rimosso dopo 5 giorni di trattamento, l’80% di petrolio emulsionato nel mezzo in uno studio del 2020, e ha ridotto in maniera efficiente i PAHs presenti in uno studio del 2013. In questo elaborato di tesi la microalga analizzata è stata Haematococcus pluvialis, nota all’ambiente scientifico per la sua capacità di produrre uno degli antiossidanti più potenti in natura, l’astaxantina (ASX) ma mai considerata per la bioremediation di idrocarburi di origine petrolifera. Lo scopo di questo progetto di dottorato è stato quello di ricercare una nuova e innovativa tecnologia che grazie all’impiego di microalghe possa rimuovere efficacemente gli idrocarburi petroliferi in matrici acquose, creando diversi genotipi in grado di agire in maniera più efficiente rispetto al genotipo non mutato (WT). Grazie a un processo di pressione selettiva si è indotto H. pluvialis a mutare e ad adattarsi ad una condizione estremamente tossica. Una volta selezionati diversi ceppi mutanti, questi sono stati seguiti nella loro crescita e nella loro capacità di produrre astaxantina, comparandoli sempre con il WT. Poiché le nostre conoscenze non permettevano di determinare se le mutazioni avvenute fossero genetiche o epigenetiche, è stata effettuata anche un’analisi genetica tramite RAPD-PCR su trascritto per evidenziare le diversità di espressione dei diversi genotipi tra di loro e con la specie non mutata, in condizioni di non stress e in generazioni successive. È stato così possibile ottenere tramite “cluster analysis” un dendrogramma che evidenziasse le distanze di espressione genetica tra essi. Questo risultato indica che la differenza di espressione in condizioni standard è ricollegabile a modificazioni genetiche alla base. Successivamente dopo aver provato diverse condizioni di stress e bioremediation, sono stati fatti crescere in presenza dell’1% di greggio puro in condizioni anaerobiche, per valutare l’attività di rimozione e/o degradazione. Le analisi chimiche effettuate solo dopo 10 giorni di trattamento hanno subito evidenziato come la quantità di carbonio presente nel mezzo di coltura era quasi raddoppiata mentre altri parametri come fluoruri, cloruri e solfati sono diminuiti. Dopo 40 giorni di trattamento, analisi preliminari tramite GS-MS e GC-FID hanno evidenziato come sia nel mezzo acquoso, che nella biomassa algale, vi erano idrocarburi e sostanze oleose, risultati confermati, poi, da un’analisi quantitativa a 20 e 160 giorni in cui si evidenzia, con diverse cinetiche, una diminuzione degli idrocarburi, che per il ceppo mutante PA1004 erano stati totalmente rimossi sia dal mezzo che dalla biomassa. Inoltre, dopo 120 giorni è stata ripetuta l’analisi genetica per valutare eventuali meccanismi adattativi avvenuti in seguito allo stress indotto dal petrolio. Mantenendo le stesse e identiche condizioni di amplificazione (primer, temperature e concentrazioni) il risultato della “cluster analysis” ha dimostrato come, effettivamente, ci fossero stati dei cambiamenti a livello di espressione genetica, portando a una riorganizzazione delle distanze ottenute precedentemente in condizioni ottimali. Queste distanze rispecchiano il comportamento dei genotipi in presenza dell’agente esogeno. Alcuni hanno rimosso totalmente il petrolio, altri hanno agito solo sulla frazione oleosa contribuendo a creare un particolato bituminoso raggruppandosi come da dendrogramma generato. L’assenza di idrocarburi all’interno del mezzo acquoso e della biomassa algale costituisce di per sé un risultato estremamente interessante andando a confermare gli obiettivi preposti all’inizio di questo percorso di ricerca. Non solo H. pluvialis è stato in grado di sopravvivere in presenza di petrolio greggio, ma anche i genotipi generati, sono risultati essere una promettente alternativa nella bioremediation degli idrocarburi. In futuro ci si predispone ad analizzare non solo le pathways coinvolte dalle mutazioni, per comprendere i meccanismi sottesi a tale processo, ma anche analizzare i sottoprodotti generati per riutilizzarli in un’ottica di economia circolare atta alla rimozione e al riutilizzo di un prodotto potenzialmente dannoso per l’uomo e l’ambiente

Bioremediation of Crude Oil by Haematococcus Pluvialis: A Preliminary Study

Nowadays, oil pollution is one of the main environmental problems. The current methods for recovering spills mainly involve chemical agents, but scientific research has focused on more natural and less harmful techniques for the environment, including a consortium of bacteria and microalgae to clean up water contaminated by hydrocarbons. The purpose of this preliminary study was to evaluate the ability of a microalga belonging to Chlorophyceae to grow in the presence of crude oil and remove the principal contaminants. H. pluvialis, which is usually used for nutraceutical purposes, thanks to the production of astaxanthin, was able to grow in anaerobic conditions, varying its metabolism from autotrophic to heterotrophic, exploiting the carbon present in the solution deriving from the presence of 1% of crude oil. Furthermore, the results of bioremediation showed a relevant reduction in chemical pollutants such as nitrate, fluoride, sulfate, and phosphate. The most important aspect of the study was the reduction after 160 days in the hydrocarbon concentration inside not only the culture medium (−32%) but also the algal biomass (−80.25%), demonstrating an optimized degradation rather than a simple absorption inside the alga

Crude Oil Bioremediation: From Bacteria to Microalgae

Crude oil is one of the major pollutants present. Its extraction and processing generate processing waters contaminated by hydrocarbons which are harmful to both human health and the flora and fauna that come into contact with it. Hydrocarbon contamination can involve soil and water, and several technologies are used for recovery. The most used techniques for the recovery of spilt oil involve chemical-physical methods that can remove most of the pollutants. Among these, must consider the bioremediation by microorganisms, mostly bacterial capable of degrading many of the toxic compounds contained within the petroleum. Microalgae participate in bioremediation indirectly, supporting the growth of degrading bacteria, and directly acting on contaminants. Their direct contribution is based on the activation of various mechanisms ranging from the production of enzymes capable of degrading hydrocarbons, such as lipoxygenases, to the attack through the liberation of free radicals. The following review analyzed all the works published in the last ten years concerning the ability of microalgae to remove hydrocarbons, intending to identify in these microorganisms an alternative technology to the use of bacteria. The advantages of using microalgae concern not only their ability to remove toxic compounds and release oxygen into the atmosphere but their biomass could then be used in a circular economy process to produce biofuels

Genetic Improvement to Obtain Specialized Haematococcus pluvialis Genotypes for the Production of Carotenoids, with Particular Reference to Astaxanthin

Nowadays, the search for natural substances with a high nutraceutical effect positively impact the world market. Among the most attractive macromolecules are antioxidants, capable of preventing the development of various pathologies. Astaxanthin (ASX) is antioxidant molecule produced by the microalga H. pluvialis as a response to different types of stress. Usually, astaxanthin production involves the first phase of accumulation of the biomass of H. pluvialis (green phase), which is then stressed to stimulate the biosynthesis and accumulation of ASX (red phase). In this study, the H. pluvialis wild-type strain was subjected to random mutagenesis by UV. Among the different mutant strains obtained, only two showed interesting bio-functional characteristics, such as a good growth rate. The results demonstrated that the HM1010 mutant not only has a higher growth trend than the WT mutant but accumulates and produces ASX even in the green phase. This innovative genotype would guarantee the continuous production of ASX, not linked to the two-step process and the uniqueness of the product obtained

An innovative protocol to select the best growth phase for astaxanthin biosynthesis in H. pluvialis.

H. pluvialis is a green unicellular microalgae and it is the first producer of natural astaxanthin in the world if subjected to stress conditions such as high light, high salinity and nutrient starvation. Astaxanthin is a powerful antioxidant used in many fields, such as aquaculture, pharmaceutical, food supplements and cosmetic. To obtain a large amount of astaxanthin, researcher focused on the optimisation of H. pluvialis growth. H. pluvialis has four different size growth stage (macrozooids, microzooids, palmelloid and “red non-motile astaxanthin accumulated encysted”), and astaxanthin production occur in the last phase. Recent studies shown that non-motile cells can produce more astaxanthin than motile cells if subjected to light stress. For these reasons, the aim of this study is to find a new and innovative methodology to select and recovery H. pluvialis in his last growth phase thanks to an electrophoretic run, and optimize, in this way, astaxanthin production

Effects of extracts of two selected strains of Haematococcus pluvialis on adipocyte function

Recently, microalgae are arousing considerable interest as a source of countless molecules with potential impacts in the nutraceutical and pharmaceutical fields. Haematococcus pluvialis, also named Haematococcus lacustris, is the largest producer of astaxanthin, a carotenoid exhibiting powerful health effects, including anti-lipogenic and anti-diabetic activities. This study was carried out to investigate the properties of two selected strains of H. pluvialis (FBR1 and FBR2) on lipid metabolism, lipolysis and adipogenesis using an in vitro obesity model. FBR1 and FBR2 showed no antiproliferative effect at the lowest concentration in 3T3-L1 adipocytes. Treatment with FBR2 extract reduced lipid deposition, detected via Oil Red O staining and the immunocontent of the adipogenic proteins PPARγ, ACLY and AMPK was revealed using Western blot analysis. Extracts from both strains induced lipolysis in vitro and reduced the secretion of interleukin-6 and tumor necrosis factor-α. Moreover, the FBR1 and FBR2 extracts improved mitochondrial function, reducing the levels of mitochondrial superoxide anion radical and increasing mitochondrial mass compared to untreated adipocytes. These findings suggest that FBR2 extract, more so than FBR1, may represent a promising strategy in overweight and obesity prevention and treatment

Lipids from Microalgae for Cosmetic Applications

In recent years, there has been considerable interest in using microalgal lipids in the food, chemical, pharmaceutical, and cosmetic industries. Several microalgal species can accumulate appreciable lipid quantities and therefore are characterized as oleaginous. In cosmetic formulations, lipids and their derivatives are one of the main ingredients. Different lipid classes are great moisturizing, emollient, and softening agents, work as surfactants and emulsifiers, give consistence to products, are color and fragrance carriers, act as preservatives to maintain products integrity, and can be part of the molecules delivery system. In the past, chemicals have been widely used but today’s market and customers’ demands are oriented towards natural products. Microalgae are an extraordinary source of lipids and other many bioactive molecules. Scientists’ attention to microalgae cultivation for their industrial application is increasing. For the high costs associated, commercialization of microalgae and their products is still not very widespread. The possibility to use biomass for various industrial purposes could make microalgae more economically competitive

Immunometabolism Modulation by Extracts from Pistachio Stalks Formulated in Phospholipid Vesicles

Several studies have demonstrated the effectiveness of plant extracts against various diseases, especially skin disorders; namely, they exhibit overall protective effects. The Pistachio (Pistacia vera L.) is known for having bioactive compounds that can effectively contribute to a person’s healthy status. However, these benefits may be limited by the toxicity and low bioavailability often inherent in bioactive compounds. To overcome these problems, delivery systems, such as phospholipid vesicles, can be employed. In this study, an essential oil and a hydrolate were produced from P. vera stalks, which are usually discarded as waste. The extracts were characterized by liquid and gas chromatography coupled with mass spectrometry and formulated in phospholipid vesicles intended for skin application. Liposomes and transfersomes showed small size (<100 nm), negative charge (approximately −15 mV), and a longer storage stability for the latter. The entrapment efficiency was determined via the quantification of the major compounds identified in the extracts and was >80%. The immune-modulating activity of the extracts was assayed in macrophage cell cultures. Most interestingly, the formulation in transfersomes abolished the cytotoxicity of the essential oil while increasing its ability to inhibit inflammatory mediators via the immunometabolic citrate pathway

Nanoecology: Exploring Engineered Nanoparticles’ Impact on Soil Ecosystem Health and Biodiversity

NANOTECHNOLOGY is a growing field that explores the interactions between engineered nanoparticles and ecosystems, with a focus on soil health and biodiversity. Engineered nanoparticles are intentionally designed at the nanoscale and offer unique properties and diverse applications, making them increasingly prevalent in consumer products and industrial processes. However, their release into the environment has raised concerns about potential ecological consequences, particularly their impacts on soil health. Studies have shown that engineered nanoparticles can have complex effects on soil microbial communities and nutrient cycling, with responses ranging from positive to adverse. Additionally, their ability to be absorbed and translocated by plants brings upon questions about their potential bioaccumulation in food chains and their effects on higher trophic levels. Understanding these intricate interactions is crucial for developing sustainable nanotechnology applications that can benefit agriculture and environmental remediation without compromising the ecosystem health. Nanoecology is an emerging field that requires attention to ethical and regulatory considerations in the use of nanomaterials. To ensure that these advanced technologies contribute positively to the ecosystem, researchers and policymakers must address these aspects. By understanding the complex interactions between nanoparticles and ecosystems, nanoecology offers the potential for innovative solutions that promote sustainable coexistence between nanotechnology and the natural world. This study specifically focuses on the relationship between engineered nanoparticles and soil health profiling. It provides a concise overview of this relationship, emphasizing the importance of responsible nanoparticle use. Additionally, the study highlights the need for monitoring soil health in soils contaminated with nanoparticles. Overall, this research underscores the significance of considering ethical and regulatory factors in the use of nanomaterials. It also emphasizes the importance of understanding the impact of nanoparticles on soil health and the need for responsible practices in their application