Mikroalg ve siyanobakterilerden bazı kimyasal bileşiklerinin üretimi ekstraksiyonu saflaştırılması ve aktivite tayini

Siyanobakteriler (mavi-yeşil alg) ve mikroalgler fikobiliprotein (PBP), karotenoidler ve çoklu doymamış yağ asitleri gibi önemli yüksek değerli ürünler, sayısız önemli biyoaktif ve biyoteknolojik olarak önemli kimyasalların gıda, kozmetik, ilaç ve nutrasötik endüstrilerinde uygulanan değerli bir kaynağı oluştururlar. Bu tezin ilk bölümünde, kültür koşullarının optimizasyonu ile siyanobakteri ve mikroalglerin biyokütle ve yüksek değerli bileşiklerin (fikosiyanin ve fukoksantin) arttırılması için yanıt yüzey yöntemi (RSM) kullanılarak gerçekleştirilmiştir. RSM ile tahmin edilen Arthrospira platensis'nin maksimum biyokütle ve fikosiyanin içeriği sırasıyla 1,06 g L−1 ve 107 mg L−1 bulunmasına rağmen maksimum biyokütle ve fikosiyanin içeriği optimize edilmiş koşullar (sıcaklık 33 ± 2 ºC, ışık şiddeti 44 μmol photons m−2 s−1 ve karıştırma hızı 120 rpm) altında validasyon deneylerinde 1,32 g L−1 ve 127 mg L−1 olarak elde edilmiştir. Ayrıca, optimize edilmiş koşullarda (sıcaklık 33 ± 2 ºC, ışık şiddeti 44 μmol fotonlar m−2 s−1 ve 2,5 L dak−1 hava akış oranı) 7L Panel foto-biyoreaktörde (PPBR) Arthrospira platensis'in büyüme ve fikosiyanin içeriği üzerindeki etkisi araştırılmıştır ve PPBR'de büyüme hızı 0,202 gün−1 ve 228 mg L−1 fikosiyanin içeriği ile 2,42 g L−1 maksimum biyokütle veriminin elde edildiği gözlenmiştir. Benzer şekilde Phaeodactylum tricornutum tarafından fukoksantin üretimi için kültür koşullarının optimizasyonu yapılmış, RSM tarafından tahmin edilen maksimum fukoksantin içeriği 7,9 mg g-1 iken, maksimum fukoksantin içeriği 10,22 mg g-1 optimize edilmiş koşullar altında (sıcaklık 15 °C, ışık şiddeti 55 μmol photons m−2 s−1 ve karıştırma hızı 130 rpm) doğrulama deneylerinde elde edilmiştir. Ayrıca, optimize edilmiş koşullarda (sıcaklık 15 °C, ışık şiddeti 55 μmol photons m−2 s−1 ve 2,5 L dak−1 hava akış oranı) 7L Panel fotobiyoreaktörde (PPBR) P. tricornutum'un fukoksantin içeriği üzerindeki etkisi araştırılmıştır ve PPBR'de maksimum fukoksantin içeriği 44,13 mg g-1 elde edilmiştir. Ek olarak, farklı azot kaynaklarının büyüme ve fikobiliprotein (PBP: PC, APC ve PE) bileşimleri üzerindeki etkisi üç siyanobakteri suşunda incelenmiştir. 12 günlük kesikli üretimde, Phormidium sp. ve Pseudoscillatoria sp. test edilen tüm nitrojen kaynaklarını kullanabilmişler olmasına rağmen her iki suş için de en iyi azot kaynağı amonyum klorür ve maksimum büyüme oranları sırasıyla µ=0,284±0,03 ve µ=0,274±0,01 gün–1, fikobiliprotein içerikleri %19,38±0,09 ve %19,99±0,14 kuru ağırlık olarak belirlenmiştir. Oysaki, Arthrospira platensis için en yüksek büyüme oranı µ= 0,304 ± 0,0 gün – 1 ve fikobiliprotein içeriği %22,27 ± 0,21 kuru ağırlıkta sodyum nitratla elde edildi. Bu tezin ikinci bölümünde, çeşitli ekstraksiyon tamponlarının kullanıldığı farklı ekstraksiyon metotlarının A. platensis ve P. tricornutum'dan elde edilen fikoksiyanin ve fukoksantin ekstraksiyonu için ekstraksiyon yöntemlerinin etkinliğinin belirlenmesi değerlendirilmiştir. Bu çalışmada değerlendirilen farklı hücre parçalama yöntemleri arasından ultrasonikasyon (2 dak) ile birlikte donma-çözmede (bir kez) ekstraksiyonu tamponu olarak Na-fosfat kullanılarak yapılan fikosiyanin ekstraksiyonu ve maksismum C-PC eldesi için etkili yöntem olduğu kanıtlanmıştır. Ayrıca, fikosiyanin iki aşamalı kromatografik yöntem kullanılarak saflaştırıldı ve analitik dereceli saflıkta (A620/A280 oranı> 4) elde edilmiştir. Saflaştırılan C-fikosiyanin ve ve β alt birimlerine karşılık gelen iki bandın varlığı SDS-PAGE de gösterilmiştir. P. tricornutum'dan fukoksantin ekstraksiyonu için araştırılan farklı ekstraksiyon yöntemleri arasında ekstraksiyon çözücüsü olarak metanol kullanılarak yapılan ultrason destekli ekstraksiyon yönteminin (UAE) en etkili yöntem olduğu kanıtlanmıştır. Ekstraksiyondan sonra fukoksantin açık silika kolon kromatografisi kullanılarak başarıyla saflaştırılmıştır. Ayrıca, ham ekstraktların ve saflaştırılmış fikosiyanin ve fukoksantinin biyoaktivitesi farklı patojen bakterilere (Escherichia coli, Moraxella catarrhalis, Staphylococcus aureus ve Candida albicans) karşı değerlendirilmiştir ve aktiviteleri gözlemlenmiştir.Cyanobacteria (blue- green algae) and Microalgae have emerged as an important source of high-value products, counting important bioactive and biotechnologically significant chemicals like phycobiliprotein (PBP), carotenoids and polyunsaturated fatty acids with potential biotechnological application in food, cosmetic, pharmaceuticals and nutraceuticals industries. In the first part of this thesis, optimization of culture conditions to enhance biomass and high value added compounds (phycocyanin and fucoxanthin) of cyanobacteria and microalgae was carried out by using response surface methodology (RSM). The predicted maximum biomass and phycocyanin content of Arthrospira platensis by RSM was 1.06 g L−1 and 107 mg L−1, respectively, whereas maximum biomass and phycocyanin content of 1.32 g L−1 and 127 mg L−1 was obtained in the flask validation experiments under optimized conditions (temperature 33±2 ºC, light irradiance 44 μmol photons m−2 s−1 and agitation speed 120 rpm). Further, influence of optimized conditions (temperature 33±2 ºC, light irradiance 44 μmol photons m−2 s−1 and air flow rate of 2.5 L min−1) on growth and phycocyanin content of A. platensis in 7L Panel photobioreactor (PPBR) cultivation was investigated and it was observed that a maximum biomass yield of 2.42 g L−1 with a specific growth rate 0.202 day−1 and phycocyanin content of 228 mg L−1 was obtained in the PPBR. In case of optimization of culture conditions for fucoxanthin production by P. tricornutum, the predicted maximum fucoxanthin content by RSM was 7.9 mg g−1, whereas maximum fucoxanthin content of 10.22 mg g−1 was obtained in the flask validation experiments under optimized conditions (temperature 15°C, light irradiance 55 μmol photons m−2 s−1 and agitation speed 130 rpm). Further, influence of optimized conditions (temperature 15°C, light irradiance 55 μmol photons m−2 s−1 and air flow rate of 2.5 L min−1) on fucoxanthin content of P. tricornutum in 7L Panel photobioreactor (PPBR) cultivation was investigated and it was observed that a maximum fucoxanthin content of 44.13 mg g−1 was obtained in the PPBR. Moreover, the effect of different nitrogen sources on growth, and phycobiliprotein (PBP: PC, APC and PE) composition of three cyanobacterial strains were investigated. In the batch culture period of 12 days, Phormidium sp. and Pseudoscillatoria sp. were able to utilize all tested nitrogen sources, however, ammonium chloride was the best nitrogen source for both strains to achieve maximum growth rate µ=0.284±0.03 and µ=0.274±0.01 day–1, phycobiliprotein contents 19.38±0.09% and 19.99±0.14% of dry weight, respectively. Whereas, for Arthrospira platensis, the highest growth rate of µ=0.304±0.0 day–1 and phycobiliprotein content of 22.27±0.21% of dry weight were achieved with sodium nitrate. In the second part of this thesis, different extraction methods using various extraction buffers were evaluated to understand the efficiency of extraction methods for phycocyanin and fucoxanthin extraction from A. platensis and P. tricornutum, respectively. Among the different extraction methods evaluated in this investigation, a combined method of freeze ; thaw plus ultrasoication using Na-phosphate as an extraction buffer was proved to be an effective method for phycocyanin extraction and observed a maximum C-PC yield. Furthermore, phycocyanin was purified using two-step chromatographic method and the analytical grade purity (A620/A280 ratio > 4) was attained. SDS-PAGE demonstrated the purity and presence of two bands corresponding to α and β subunits of the C-phycocyanin. Whereas, among the different extraction methods studied for fucoxanthin extraction from P. tricornutum, Ultrasound assisted extraction method (UAE) using methanol as an extraction solvent was proved to be an effective method. Furthermore, fucoxanthin was successfully purified using open silica column chromatography. Moreover, the bioactivity of crude extracts and purified phycocyanin as well as fucoxanthin was assessed against different pathogenic bacteria and observed the activity against Escherichia coli, Moraxella catarrhalis, Staphylococcus aureus and Candida albicans

Acclimation and characterization of marine cyanobacterial strains Euryhalinema and Desertifilum for C-phycocyanin production

This study involves evaluation of two native cyanobacterial strains Euryhalinema and Desertifilum isolated from a mangrove pond in Haikou (China) for their possible phycocyanin (C-PC) production. Maximal growth rate with highest chlorophyll and C-PC accumulation were observed at 28°C and 60 μmol photons m(−2) s(−1) photon flux density for Euryhalinema sp., while for Desertifilum sp. at 32°C and 80 μmol photons m(−2) s(−1). Nitrogen and iron concentration trails revealed that double strength concentration of sodium nitrate and ferric ammonium citrate in original BG11 media increased growth rate and accumulation of C-PC for both strains. Three different C-PC extraction methods were tested. The combined extraction protocol of freeze–thaw and ultrasonication markedly increased the C-PC extraction efficiency and attained the food grade purity (A (620)/A (280) ratio >0.7), whereas a higher C-PC yield was found with Na-phosphate buffer. Furthermore, the clarified crude extract was used to purify C-PC by fractional ammonium sulfate [(NH₄)₂SO₄] precipitation, Sephadex G-25 gel filtration chromatography, and DEAE-sephadex ion exchange chromatography and attained analytical grade purity (A (620)/A (280) ratio >3.9). Taken together, both strains showed their potential to be domesticated for valuable phycocyanin production

Analytical Grade Purification of Phycocyanin from Cyanobacteria

Phycocyanin is a blue-colored pigment-protein complex that exhibits numerous biofunctions such as anti-inflammation, antioxidation, antitumor, neuroprotective effect, and immunological enhancement. Purified phycocyanin has pharmaceutical and nutraceutical applications. In addition, as a nontoxic and non-carcinogenic natural coloring agent, phycocyanin has many applications in the food and cosmetic industries. This chapter describes a protocol for extraction and analytical grade purification of phycocyanin from cyanobacteria. The purification steps include (1) extraction of phycocyanin from biomass, (2) ammonium sulfate precipitation of phycocyanin and dialysis, and (3) purification of phycocyanin by gel filtration and ion-exchange chromatography

Characterization of different chitosanases of Bacillus strains and their application in chitooligosaccharides production

Chitosanases are potential candidates for chitooligosaccharides (COS) production-based industries, therefore, the discovery of chitosanases having commercial potential will remain a priority worldwide. This study aims to characterize different chitosanases of Bacillus strains for COS production. Six different indigenous Bacillus strains (B. cereus EGE-B-6.1m, B. cereus EGE-B-2.5m, B. cereus EGE-B-5.5m, B. cereus EGE-B-10.4i, B. thuringiensis EGE-B-3.5m, and B. mojavensis EGE-B-5.2i) were used to purify and characterize chitosanases. All purified chitosanases have a similar molecular weight (37 kDa) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, other characteristics such as optimum temperature and pH, kinetic parameters (K-m and V-max), temperature, and pH stabilities were dissimilar among the strains of different Bacillus species and within the same species. Furthermore, chitosanases of all strains were able to successfully hydrolyze chitosan to COS and oligomers of the degree of polymerization 2-6 were detected with chitobiose and chitotriose as major hydrolysis products. The relative yields of COS were in a range of 19%-31% and chitosanase of B. thuringiensis EGE-B-3.5m turned out to be the best enzyme in terms of its characteristics and COS production potential with maximum relative yield (31%). Results revealed that Bacillus chitosanases could be used directly for efficient bioconversion of chitosan into COS and will be valuable for large-scale production of biologically active COS.Ege University Scientific Research Projects Coordination Unit [15FBE-11]This study was supported by Ege University Scientific Research Projects Coordination Unit (Project No. 15FBE-11)

Data_Sheet_1_Natural deep eutectic solvents-based green extraction of vanillin: optimization, purification, and bioactivity assessment.docx

The sustainable extraction of natural compounds has recently attracted significant attention. The extraction of high-quality natural vanillin in active form is crucial for its efficient use in various industries, but conventional solvents are not suitable for this purpose. The flammability, volatility, and toxicity of organic solvents can harm extraction personnel, and their waste liquid can cause environmental pollution. Natural deep eutectic solvents (NADES) are cost-effective, environmentally friendly, biodegradable, and non-toxic organic alternative to conventional solvents. In this study, 20 different NADES were tested for the sustainable extraction of natural vanillin. Among these, a DES system composed of choline chloride: 1,4-butanediol: lactic acid exhibited the highest extraction rate (15.9 mg/g). Employing response surface methodology (RSM), optimal extraction conditions were determined, yielding a vanillin content 18.5 mg/g with water content of 33.9%, extraction temperature of 64.6°C, extraction time of 32.3 min, and a solid-liquid ratio of 44.9 mg/mL. Subsequently, the optimized NADES system was then assessed for reusability in extracting vanillin from vanilla pods and kraft lignin over three cycles, retaining 43% of its extraction efficiency and demonstrating potential for waste reduction. Purification of vanillin was achieved through chromatography using a non-polar resin SP700, with ethanol as a desorption eluent and a feed solution pH of 4.0, resulting in the highest vanillin purity. HPLC and GC-MS analyses confirmed purity, while antioxidant activity assays (DPPH and ABTS) showcased significant antioxidant activity of the purified vanillin. Moreover, vanillin exhibited notable antimicrobial activity against a panel of food-borne bacteria. This study introduces an environmentally friendly approach to vanillin extraction highlights using NADES, emphasizing the potential for producing high-quality bioactive vanillin with reduced environmental impact. The applicability of NADES systems extends beyond vanillin, offering a versatile method for extracting diverse natural compounds.</p

Mixotrophic cultivation of microalgae for carotenoid production

The intrinsic ability of microalgae to accumulate high amounts of carotenoids has made them the preferred aquatic organisms of biotechnological exploration for carotenoid production. To continuously innovate and modify microalgal bioprocesses, aquaculture scientists have been working hard for the past decades in a forwardlooking way, and mixotrophic cultivation of microalgae is deemed as a promising strategy to decrease production cost. This review is intended to summarise the recent research advancement of carotenoids production from mixotrophically cultivated microalgae, starting from the structure, biosynthesis, physiological roles and applications of carotenoids and followed by the production processes both currently established and under development. Most importantly, the microalgal physiology of mixotrophic cultivation is reviewed in depth both in general and specifically for the most studied species, and the prospects of commercially viable mixotrophic microalgal processes for carotenoid production along with the insight of future research are of course discussed. Finally, we conclude that mixotrophy might be a promising strategy for large-scale cultivation of microalgae to produce carotenoids although some technical obstacles need to be overcome