The influence of different flocculants on the physiological activity and fucoxanthin production of Phaeodactylum tricornutum

Phaeodactylum tricornutum is an economically important species of microalgae that is widely used in aquaculture, and it is rich in bioactive substances including eicosapentaenoic acid and fucoxanthin. The major bottleneck for industrialization of this species is harvesting. Flocculation is used to harvest microalgae, thus the selection of flocculants is of great importance. In this study, we compared the flocculation effect of four different chemicals (ferric chloride, aluminum sulphate, polyaluminum chloride, and aluminum potassium sulphate) on P. tricornutum. Microexamination showed that ferric and aluminum salts had similar flocculation effects on the algae. Growth and chlorophyll fluorescence measurements showed that P. tricornutum can be re-cultured after flocculation. Pigment analysis showed that flocculation did not result in degradation of fucoxanthin, which suggests that the four flocculants tested may be useful for industrial applications. The results also showed that ferric chloride was the best flocculant for harvesting P. tricornutum when the target product was fucoxanthin, as it had the least influence on the physiological activity of P. tricornutum and it did not lead to degradation of cell components. In contrast, aluminum is poisonous to the nervous system of animals and humans. In addition, the culture medium can be recycled after flocculation by ferric chloride.     

Optimization of Chlorella biomass harvesting by flocculation and its potential for biofuel production

Optimization of microalgal biomass harvesting is essential to produce effective and optimum outcomes that can contribute towards a feasible and economical harvesting technique. Two Chlorella species were used, namely, C. vulgaris and C. sorokiniana UKM3. Two essential factors affecting microalgal biomass harvesting via flocculation, namely, the initial pH of the microalgal broth and flocculant (chitosan) concentration were studied. The optimization process was conducted by using a response surface methodology (RSM) based on the model of face-centered-central composite design (FC-CCD). The potential for biofuel application of the harvested biomass was evaluated based on the production of fatty acid methyl esters (FAMEs) by transesterification. The quadratic models obtained from the RSM significantly fitted the experiment data as the p-values were less than 0.05. The initial pH of the microalgal suspension was found to have a more significant effect on the flocculation process than flocculant concentration. For C. vulgaris, the highest flocculant efficiency of 98.7% was obtained at a chitosan concentration of 0.2 g L^?1 and an initial pH of 12.0, whereas for C. sorokiniana UKM3, at 0.15 g L^?1 of chitosan and initial pH of 12.0 produced the highest efficiency of 97.1%. The harvested biomass of both species exhibited a high content of palmitic acid (C16:0) with 29.74 wt% and 11.81 wt% of dry biomass for C. vulgaris and C. sorokiniana UKM3, respectively. This study showed that Chlorella species can be harvested efficiently using the flocculation process and manifested an excellent potential for biodiesel production where palmitic acid (C16:0) is one of the main compounds for high-acid oil-biodiesel.

     

Optimization of pH induced flocculation of marine and freshwater microalgae via central composite design

Microalgae harvesting via pH induced flocculation along with utilization of recovered medium after flocculation is one of the most economical methods for separating the microalgal biomass in order to reduce the dewatering cost. In this study, optimization of marine and freshwater microalgae flocculation by pH adjustment was investigated via central composite design methodology. One molar of KOH and NaOH solutions were used to increase the pH level of the microalgal culture. Increasing pH value of the medium provided the highest flocculation efficiency up to 92.63 and 86.18% with pH adjusted to 10.5 with KOH and NaOH solutions for marine microalgae Nannochloropsis oculata and freshwater microalgae Chlorella minutissima, respectively. Also, it was revealed that microalgae cells were still alive after flocculation process and their biochemical composition was not changed, and flocculated medium can be used again for the next microalgal production. According to the results, it can be said that this method is cheap and effective, simple to operate and provides the utilization of flocculated medium again.     

Evaluation of Moringa oleifera seed flour as a flocculating agent for potential biodiesel producer microalgae

Microalgal biofuel alternatives have been hindered by their cost and energy intensive production. In the microalgal harvesting process, the intermediate step of flocculation shows potential in drastically reducing the need for costly centrifugation processes. Moringa oleifera seeds, which have been used for water treatment due to their high flocculation potential, low cost and low toxicity, are presented in this paper as strong candidate for flocculating Chlorella vulgaris, a microalgae with high biodiesel production potential. Early results of our group showed a very high flocculation (around 85% of biomass recovery). The aim of this work was to investigate the influence of Moringa oleifera seed flour concentration, sedimentation time and pH on the flocculation efficiency. Cell suspensions treated with Moringa seed flour (1 g L^-1) had their flocculation significantly increased with the rise of pH, reaching 89% of flocculation in 120 min at pH 9.2. Sedimentation time of 120 min and a concentration of 0.6 g L^-1 proved to be ample for substantial flocculation efficiency. In spite of the need for more research to ensure the economic viability and sustainability of this process, these results corroborate Moringa oleifera seeds as a strong candidate as a bioflocculant for Chlorella vulgaris cells and indicate optimal pH range of its action.     

Tris not only controls the pH in microalgal cultures,but also feeds bacteria

Tris (Tris(hydroxymethyl)amino methane), a compound often used as a buffer in microalgal culture media, sustains active bacterial growth in non-axenic microalgal cultures when sodium phosphate is present. The low pH levels caused by bacterial growth and probably the depletion of phosphorus in the medium caused the collapse ofPhaeodactylum tricornutum cultures resulting in a reduction of microalgal growth from 32 x 10⁶ to 1.1 x 10⁶ cells ml^–1. This emphasizes the need for care when interpreting the results of non-axenic microalgae cultures in which Tris or other organic buffer is added.     

Antioxidant potential of microalgae in relation to their phenolic and carotenoid content

In the past decades, food scientists have been searching for natural alternatives to replace synthetic antioxidants. In order to evaluate the potential of microalgae as new source of safe antioxidants, 32 microalgal biomass samples were screened for their antioxidant capacity using three antioxidant assays, and both total phenolic content and carotenoid content were measured. Microalgae were extracted using a one-step extraction with ethanol/water, and alternatively, a three-step fractionation procedure using successively hexane, ethyl acetate, and water. Antioxidant activity of the extracts varied strongly between species and further depended on growth conditions and the solvent used for extraction. It was found that industrially cultivated samples of Tetraselmis suecica, Botryococcus braunii, Neochloris oleoabundans, Isochrysis sp., Chlorella vulgaris, and Phaeodactylum tricornutum possessed the highest antioxidant capacities in this study and thus could be a potential new source of natural antioxidants. The results from the different types of extracts clearly indicated that next to the well-studied carotenoids, phenolic compounds also contribute significantly to the antioxidant capacity of microalgae.     

Modeling of microalgal shear‐induced flocculation and sedimentation using a coupled CFD‐population balance approach

In this study, shear‐induced flocculation modeling of Chlorella sp. microalgae was conducted by combination of population balance modeling and CFD. The inhomogeneous Multiple Size Group (MUSIG) and the Euler–Euler two fluid models were coupled via Ansys‐CFX‐15 software package to achieve both fluid and particle dynamics during the flocculation. For the first time, a detailed model was proposed to calculate the collision frequency and breakage rate during the microalgae flocculation by means of the response surface methodology as a tool for optimization. The particle size distribution resulted from the model was in good agreement with that of the jar test experiment. Furthermore, the subsequent sedimentation step was also examined by removing the shear rate in both simulations and experiments. Consequently, variation in the shear rate and its effects on the flocculation behavior, sedimentation rate and recovery efficiency were evaluated. Results indicate that flocculation of Chlorella sp. microalgae under shear rates of 37, 182, and 387 s^?1 is a promising method of pre‐concentration which guarantees the cost efficiency of the subsequent harvesting process by recovering more than 90% of the biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:160–174, 2018     

Anaerobic digestion of microalgal biomass with ultrasonic disintegration

The use of photosynthetic microalgae for nutrient removal and biofuel production has been widely discussed. Anaerobic digestion of waste microalgal biomass to produce biogas is a promising technology for bioenergy production. However, the methane yield from this anaerobic process was limited because of the hard cell wall of Chlorella vulgaris. The use of ultrasound has proven to be successful at improving the disintegration and anaerobic biodegradability of Chlorella vulgaris. Ultrasonic pretreatment in the range of 5–200 J ml⁻¹ was applied to waste microalgal biomass, which was then used for batch digestion. Ultrasound techniques were successful and showed higher soluble COD at higher applied energy. During batch digestion, cell disintegration due to ultrasound increased in terms of specific biogas production and the degradation rate. Compared to the untreated sample, the specific biogas production was increased in the ultrasound-treated sample by 90% at an energy dose of 200 J ml⁻¹. For the disintegrated samples, volatile solids reduction was also increased according to the energy input and degradation. These results indicate that the hydrolysis of microalgal cells is the rate-limiting step in the anaerobic digestion of microalgal biomass.     

THE EFFECT OF SODIUM CHLORIDE ON CARBONIC ANHYDRASE ACTIVITY IN MARINE MICROALGAE1

The effects of NaCl on the internal and external carbonic anhydrase (CA) activity of several marine microalgae were studied. Unlike freshwater microalgae in which CA activity is generally inhibited by NaCl, marine microalgae exhibited considerable species-dependent variation when exposed to NaCl. CA activity in Phaeodactylum tricornutum, a diatom, was inhibited, whereas it was activated in the coccolithophorid Pleurochrysis carterae. CA activity in the chlorophyte Dunaliella primolecta was not significantly affected by NaCl. In Dunaliella salina and Dunaliella parva, NaCl inhibited external CA without affecting the internal activity, whereas in Chlorella vulgaris C-133 and Dunaliella peircei only the internal CA was inhibited. Internal CA of Dunaliella tertiolecta was not affected by NaCl, but the external enzyme was significantly enhanced. Salt substitution experiments revealed that chloride (Cl^-) is the ion affecting CA activity; the effect of Cl^- can be replaced by bromide ion. Cl^- affects external CA activity while also affecting the apparent affinity for inorganic carbon during photosynthesis. Microalgae whose internal CA activity was enhanced by Cl^- showed higher intracellular Cl^- concentrations than those species that were inhibited.     

Inducing autoflocculation in the diatom <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> through CO<Subscript>2</Subscript> regulation

The effect of pH on flocculation was studied using the diatom Phaeodactylum tricornutum and the green algae Scenedesmus cf. obliquus as surrogate species. There was a distinct, species-specific threshold of pH where flocculation started. P. tricornutum started to flocculate at pH 10.5 and S. cf. obliquus at pH 11.3. Above this threshold, settling rates up to 360 cm h⁻¹ were observed for P. tricornutum and the concentrating factor was up to 60-fold. The combined effect of pH, turbulence, and cell density on flocculation of P. tricornutum was additionally studied in a factorial 5³-design experiment. pH was the most important factor affecting flocculation, but at the pH threshold (pH 10.5), the concentrating factor was increased by increasing cell density and turbulence. Algae increases the pH during photosynthesis, and the P. tricornutum and S. cf. obliquus cultures increased the pH to a maximum of 10.8 and 9.5, respectively, after discontinuing the CO₂ supply. For P. tricornutum, this was above the flocculation threshold, and rapid settling of this species due to increased pH was observed in a matter of hours after the CO₂ supply was turned off. This could be used as a simple, low-cost, initial dewatering step for this species.     

Effects of green LED light and three stresses on biomass and lipid accumulation with two-phase culture of microalgae

The effects of wavelengths of light-emitting diode (LED), nitrate concentration, and salt concentration were evaluated for the two-phase culture of the microalgal species Phaeodactylum tricornutum, Dunaliella tertiolecta, and Isochrysis galbana on cell growth and lipid production. Blue LEDs produced the highest biomass of P. tricornutum at a nitrate concentration of 8 mg/L, reaching 0.97 g dcw/L with a specific growth rate (μ) of 0.047 h⁻¹, followed by I. galbana with 0.79 g dcw/L and μ = 0.040 h⁻¹ and D. tertiolecta with 0.55 g dcw/L and μ = 0.028 h⁻¹. Of the three microalgae, P. tricornutum had the highest specific growth rate of μ_max = 0.070 h⁻¹ and lowest saturation constant of K_s = 4.18 mg/L, resulting in fast cell growth. The highest lipid production was obtained under green LED wavelength stress on day 14, reaching 60.6% (w/w) of the dry cell weight among the three microalgae. The main fatty acids produced by the three microalgae were myristic acid (C14:0), palmitic acid (C16:0), oleic acid (C18:1), and arachidic acid (C20:0), which comprised 72.68%–84.16% (w/w) of the total fatty acids content under three stresses.     

Biodegradation of p-nitrophenol by microalgae

A study was made on the use of a mixed microalgal consortium to degrade p-nitrophenol. The consortium was obtained from a microbial community in a waste container fed with the remains and by-products of medium culture containing substituted aromatic pollutants (nitrophenols, chlorophenols, fluorobenzene). After selective enrichment with p-nitrophenol (p-NP), followed by an antibiotic treatment, an axenic microalgal consortium was recovered, which was able to degrade p-nitrophenol. At a concentration of 50 mg L^–1, total degradation occurred within 5 days. Two species, Chlorella vulgaris var. vulgaris f. minuscula and Coenochloris pyrenoidosa, were isolated from the microalgal consortium. The species were able to accomplish p-NP biodegradation when cultured separately, although Coenochloris pyrenoidosa was more efficient, achieving the same degradation rate as the original axenic microalgal consortium. When Coenochloris pyrenoidosa was associated with Chlorella vulgaris in a 3:1 ratio, complete removal of the nitro-aromatic compound occurred within three days. This is apparently the first report on the degradation of a nitro-aromatic compound by microalgae.     

The effect of pH in intensive microalgal cultures. II. Species competition

The results of a series of competition experiments between the chlorophyte Dunaliellatertiolecta (Dun) Butcher and the diatom Phaeodactylum tricornutum (TFX-1) Bohlin demonstrate conclusively that Phaeodactylum tricornutum dominates in intensive marine cultures when the pH rises above ≈ 10. This dominance results because of the diatom's unique ability among marine species to tolerate alkaline conditions. When the pH is regulated both freshwater and marine algae, once firmly established in culture at their respective pH optima, can resist invasion from competing species. Hence. pH control may be a method for maintaining species other than P. tricornutum in mass culture. When Dunaliella tertiolecta, however, is grown under even slight pH stress it becomes susceptible to invasion by Phaeodactylum tricornutum. This susceptibility to takeover by P. tricornutum increases with increasing pH. In contrast, the freshwater chlorophyte Chlorella vulgaris Beij., which also is sensitive to increasing pH, is capable of remaining dominant at any pH within its tolerance range when invaded by the pH-insensitive chlorophyte, Scenedesmus obliquus (Turp.) Kutz. Although allelopathic interactions may be responsible for the success of Chlorella vulgaris under seemingly stressful conditions, the success of Phaeodactylum tricornutum at increasingly higher pH seems to be related primarily to the alga's pH tolerance characteristics and not to any chemical interactions with competing species.     

Harvesting of Chlorella vulgaris using a bioflocculant from Paenibacillus sp. AM49

Microbial flocculants for harvesting mass cultured Chlorella vulgaris were screened and that from Paenibacillussp. AM49 was identified as the best. The flocculation efficiency of this bioflocculant increased with the pH within a range of pH 5–11 and was 83%, which was higher than the 72% and 78% produced by aluminum sulfate and polyacrylamide, respectively. The highest flocculation efficiency was with 6.8 mm CaCl₂ as co-flocculant. The bioflocculant from Paenibacillussp. AM49 can be used effectively to harvest C. vulgaris from large-scale cultures.     

Effects of outdoor cultures on the growth and lipid production of <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> using closed photobioreactors

One of the principal challenges for large scale production of microalgae is the high costs of biomass production. Aiming for minimize this problem, microalgal biodiesel production should focus on outdoors cultures, using available solar light and allowing lower energy cost process. Testing species that proved to be common and easy to culture may be a good approach in this process. The present work reports indoor-outdoor cultures of Phaeodactylum tricornutum using different bioreactors types, using cell growth, biochemical composition, and the profiles of the fatty acids produced as the parameters to test the optimization processes. The results show that the use of outdoor cultures is a good choice to obtain P. tricornutum biomass with a good potential for biodiesel production. The microalgae produced reached better growth efficiency, major lipid content and showed an increment in the percentage of saturated fatty acids (required on the biodiesel production) respect indoor cultures. These results are important to show the relevance of using outdoor cultures as a way to improve the efficiency and the energetic balance of the biodiesel production with P. tricornutum algae.     

Heavy metal sorption by microalgae

Viable microalgae are known to be able to accumulate heavy metals (bioaccumulation). Against a background of the increasing environmental risks caused by heavy metals, the microalgae Chlorella vulgaris and Spirulina platensis and their potential for the biological removal of heavy metals from aqueous solutions were taken as an example for investigation. Small-scale cultivation tests (50 1) with Cd-resistant cells of Chlorella vulgaris have shown that approx. 40% of the added 10 mg Cd/l was removed from the solution within seven days. At this heavy metal concentration sensitive cells died. Non-viable microalgae are able to eliminate heavy metal ions in a short time by biosorption in uncomplicated systems, without any toxicity problems. Compared with original biomasses, the sorption capacity of microalgal by-products changes only insignificantly. Their low price makes them economical.     

Harvesting <Emphasis Type="Italic">Chlorella minutissima</Emphasis> using cell coagulants

Twelve salts were tested for their ability to coagulate microalgae cells in cultures of Chlorella minutissima. The final aim was to develop an easy and efficient approach for harvesting microalgae biomass in dense cultures. Aluminum, ferric, and zinc salts coagulated C. minutissima cultures, while optimum concentration was 0.75 and 0.5 g L⁻¹ for sulfate and chloride salts, respectively. Aluminum salts were most efficient, but caused some cell lysis, which may render this approach inappropriate in some cases. Ferric and zinc salts were ranked second and third, respectively, according to their culture cell-coagulation efficiency. Ferric salts caused a change in the color of the cells, mainly at concentrations higher than 1 g L⁻¹. Zinc salts were less harmful for the microalgal cells, but an additional problem was observed with cell aggregates adhering to the walls of the glass test tubes. Selection of the appropriate coagulant is related to the purpose of the coagulation process.     

Monitoring microalgal growth of Chlorella minutissima with a new all solid-state contact nitrate selective sensor

As third generation feedstock, microalgae are microorganisms that can grow only in the optimum conditions. There are parameters including the concentration of macro and microelements in nutrient solution, pH, temperature and light intensity that have significant impact on microalgal growth. In recent years, various sensing devices have been developed for sensitive measurement of these parameters during microalgal growth. In this study, a new potentiometric nitrate selective sensor was developed to indicate the nitrate uptake of microalgae and the effect of nitrate nutrient on microalgal growth, specifically, and this sensor was successfully applied to determine nitrate concentration in medium during microalgal growth. Moreover, the effects of nitrate, carbonate and phosphate concentration in the growth medium on biomass production of Chlorella minutissima were investigated by using Box–Behnken design method, and optimum conditions were determined for the highest biomass production of microalgae. As a result of the experiments, it was seen that the highest C. minutissima production was achieved using the medium consist of 2.63 g/L NaNO₃, 0.35 g/L Na₂CO₃ and 0.4 g/L KH₂PO_4. Statistically, it was observed that there was a proportional relationship between the microalgae production and investigated parameters such as carbon, nitrogen and phosphate amounts of culture mediums. The electrode showed a wide linear range between 1.0 × 10⁻¹ and 5.0 × 10⁻⁵ M with a detection limit of the 5 × 10⁻⁶ M and the response time was found as 10 s. The results showed that developed nitrate selective sensor could be successfully applied for continuous measurement of nitrate in microalgal productions at reduced cost.     

Growth inhibition of the eight species of microalgae by growth inhibitor from the culture of Isochrysis galbana and its isolation and identification

A type of growth inhibitor was successfully isolated and purified from cell-free filtrates of cultural medium at the death phase of Isochrysis galbana, and its chemical structure was confirmed by the methods of FABMS, UV, ¹H-NMR, ¹³C-NMR and 2D NMR, which was 1-[hydroxyl-diethyl malonate]-isopropyl dodecenoic acid, C₂₂H₃₈O₇. The results showed that the growth-inhibitor strongly inhibited the growth of Isochrysis galbana, and the growth of the eight species of microalgae (Dunaliella salina, Platymonas elliptica, Chlorella vugralis, Nitzschia closterium, Chaetoceros muelleri, Chaetoceros gracilis, Nitzschia closterium minutissima, Phaeodactylum tricornutum) also could be regulated by the growth-inhibitor in a concentration-dependent manner. The further investigation found that the synthesis process of chlorophyll and protein in the cells of all test microalgae could be inhibited by the growth inhibitor, and the content of chlorophyll and protein significantly decreased.     

Potentiation of concurrent expression of lipogenic genes by novel strong promoters in the oleaginous microalga Phaeodactylum tricornutum

There has been growing interest in using microalgae as production hosts for a wide range of value-added compounds. However, microalgal genetic improvement is impeded by lack of genetic tools to concurrently control multiple genes. Here, we identified two novel strong promoters, designated Pt202 and Pt667, and delineated their potential role on simultaneously driving the expression of key lipogenic genes in Phaeodactylum tricornutum. In silico analyses of the identified promoter sequences predicted the presence of essential core cis elements such as TATA and CAAT boxes. Regulatory role of the promoters was preliminarily assessed by using GUS reporter which demonstrated strong GUS expression. Thereafter, two key lipogenic genes including malic enzyme (PtME) and 5-desaturase (PtD5b), were overexpressed by the two promoters Pt202 and Pt667, respectively, in P. tricornutum. Combinatorial gene overexpression did not impair general physiological performance, meanwhile neutral lipid content was remarkably increased by 2.4-fold. GC-MS analysis of fatty acid methyl esters revealed that eicosapentaenoic acid (EPA; C20:5) was increased significantly. The findings augment a crucial kit to microalgal genetic tools that could facilitate the multiple-gene expression driven by various promoters, and promote microalgae for industrial bioproduction.