Isolation of Novel Microalgae from Acid Mine Drainage and Its Potential Application for Biodiesel Production

Microalgae were selected and isolated from acid mine drainage in order to find microalgae species which could be cultivated in low pH condition. In the present investigation, 30 microalgae were isolated from ten locations of acid mine drainage in South Korea. Four microalgae were selected based on their growth rate, morphology, and identified as strains of KGE1, KGE3, KGE4, and KGE7. The dry biomass of microalgae species ranged between 1 and 2 g L^?1 after 21 days of cultivation. The growth kinetics of microalgae was well described by logistic growth model. Among these, KGE7 has the highest biomass production (2.05?±?0.35 g L^?1), lipid productivity (0.82?±?0.14 g L^?1), and C16–C18 fatty acid contents (97.6 %). These results suggest that Scenedesmus sp. KGE 7 can be utilized for biodiesel production based on its high biomass and lipid productivity.     

Screening of <Emphasis Type="Italic">Isochrysis</Emphasis> Strains and Utilization of a Two-Stage Outdoor Cultivation Strategy for Algal Biomass and Lipid Production

Isochrysis is a genus of marine algae without cell wall and capable of accumulating lipids. In this study, the lipid production potential of Isochrysis was assessed by comparing 15 Isochrysis strains with respect to their growth rate, lipid production, and fatty acid profiles. Three best strains were selected (lipid productivity, 103.0~121.7 mg L^?1 day^?1) and their lipid-producing capacities were further examined under different controlled parameters, e.g., growth phase, medium nutrient, and light intensity in laboratory cultures. Furthermore, the three Isochrysis strains were monitored in outdoor panel photobioreactors with various initial cell densities and optical paths, and the strain CS177 demonstrated the superior potential for outdoor cultivation. A two-stage semi-continuous strategy for CS177 was subsequently developed, where high productivities of biomass (1.1 g L^?1 day^?1) and lipid (0.35 g L^?1 day^?1) were achieved. This is a comprehensive study to evaluate the lipid-producing capability of Isochrysis strains under both indoor and outdoor conditions. Results of the present work lay a solid foundation for the physiological and biochemical responses of Isochrysis to various conditions, shedding light on the future utilization of this cell wall-lacking marine alga for biofuel production.     

Semi-continuous Cultivation of Haematococcus pluvialis for Commercial Production

The objectives of the present study on the growth of Haematococcus pluvialis were to indicate the effects of a long-term semi-continuous cultivation, sterilization, carbon dioxide, and different culture media by using artesian well water. This investigation was an enterprise in order to commercialize the production economically. When the effect of CO₂ was investigated in basal culture medium, the influence of sterilization was also researched in Rudic’s culture medium in vertical panel-type photobioreactors for 31 days of semi-continuous cultivation. The maximum cell concentration of 10.55?×?10⁵ cells ml^?1, which corresponds to the growth rate of 0.271 day^?1 with the areal productivity of 3.531 g m^?2 day^?1, was found in non-sterilized RM medium on the 24th day of the third run of semi-continuous cultivation at a renewal rate of 50% in a vertical panel-type photobioreactor.     

Growth of Cyanobacteria: Optimization for Increased Carbohydrate Content

Growths of Lyngbya limnetica and Oscillatoria obscura were investigated at varying pH, light intensity, temperature, and trace element concentration with a view to optimize these parameters for obtaining the maximum carbohydrate content. The maximum growth for both strains was obtained at pH 9.0 and temperature 20 ± 3 °C using a light intensity of 68.0 μmol m^?2 s^?1 with continuous shaking. Growth under the nitrogen starvation condition affected the carbohydrate content more compared to the phosphorus starvation, and maximum concentrations were found as 0.660 and 0.621 g/g of dry biomass for L. limnetica and O. obscura, respectively. Under the optimized nitrogen-rich conditions, the specific growth rates for the two strains were found to be 0.187 and 0.215 day^?1, respectively. The two-stage growth studies under nitrogen-rich (stage I) followed by nitrogen starvation (stage II) conditions were performed, and maximum biomass and carbohydrate productivity were found as 0.088 and 0.423 g L^?1 day^?1 for L. limnetica. This is the first ever attempt to evaluate and optimize various parameters affecting the growth of cyanobacterial biomass of L. limnetica and O. obscura as well as their carbohydrate contents.     

Cultivation of Chlorella vulgaris in Dairy Wastewater Pretreated by UV Irradiation and Sodium Hypochlorite

There is potential in the utilization of microalgae for the purification of wastewater as well as recycling the resource in the wastewater to produce biodiesel. The large-scale cultivation of microalgae requires pretreatment of the wastewater to eliminate bacteria and protozoa. This procedure is costly and complex. In this study, two methods of pretreatment, UV irradiation, and sodium hypochlorite (NaClO), in various doses and concentrations, were tested in the dairy wastewater. Combining the efficiency of biodiesel production, we proposed to treat the dairy wastewater with NaClO in the concentration of 30 ppm. In this condition, The highest biomass productivity and lipid productivity of Chlorella vulgaris reached 0.450 g L^?1 day^?1 and 51 mg L^?1 day^?1 after a 4-day cultivation in the dairy wastewater, respectively.     

Growth of Chlorella vulgaris on Sugarcane Vinasse: The Effect of Anaerobic Digestion Pretreatment

Microalgae farming has been identified as the most eco-sustainable solution for producing biodiesel. However, the operation of full-scale plants is still limited by costs and the utilization of industrial and/or domestic wastes can significantly improve economic profits. Several waste effluents are valuable sources of nutrients for the cultivation of microalgae. Ethanol production from sugarcane, for instance, generates significant amounts of organically rich effluent, the vinasse. After anaerobic digestion treatment, nutrient remaining in such an effluent can be used to grow microalgae. This research aimed to testing the potential of the anaerobic treated vinasse as an alternative source of nutrients for culturing microalgae with the goal of supplying the biodiesel industrial chain with algal biomass and oil. The anaerobic process treating vinasse reached a steady state at about 17 batch cycles of 24 h producing about 0.116 m³CH₄ kgCOD_vinasse ^?1. The highest productivity of Chlorella vulgaris biomass (70 mg l^?1 day^?1) was observed when using medium prepared with the anaerobic digester effluent. Lipid productivity varied from 0.5 to 17 mg l^?1 day^?1. Thus, the results show that it is possible to integrate the culturing of microalgae with the sugarcane industry by means of anaerobic digestion of the vinasse. There is also the advantageous possibility of using by-products of the anaerobic digestion such as methane and CO₂ for sustaining the system with energy and carbon source, respectively.     

Investigation of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> UTEX 265 Cultivation under Light and Low Temperature Stressed Conditions for Lutein Production in Flasks and the Coiled Tree Photo-Bioreactor (CTPBR)

Lutein has an increasing share in the pharmaceutical and nutraceutical market due to its benefits to eye health. Microalgae may be a potential source for lutein production while the expense limits the commercialization. In this study, a coiled tubular tree photobioreactor (CTPBR) design was investigated for cultivating the cold tolerant microalgae Chlorella vulgaris UTEX 265 under various conditions for lutein production. The influence and interaction of light irradiance strength, lighting cycle, and temperature on microalgae and lutein production efficiency at low temperature range were also studied in flasks via response surface method (RSM). The results demonstrated that 14 h day-light, 120 μmol photons m^?2 s^?1, and 10 °C was the optimal condition for algae growth and lutein production at low temperature experimental ranges. C. vulgaris UTEX 265 showed good potential to produce lutein in cold weather, and the optimum lutein production was contrary to the specific lutein content but corresponds to the trend of optimum growth. Additionally, fast growth (μ = 1.50 day^?1) and good lutein recovery (11.98 mg g^?1 day^?1) in CTPBR were also achieved at the low irradiance stress condition and the low temperature photo-inhibition conditions.     

Fatty Acid Content and Profile of the Aerial Microalga Coccomyxa sp. Isolated from Dry Environments

Aerial algae are considered to be highly tolerant of and adaptable to severe conditions including radiation, desiccation, high temperatures, and nutrient deficiency, compared with those from aquatic habitats. There are considerable variations in the fatty acid (FA) composition of aerial microalgae from dry environments. A new species with a high lipid level was found on concrete surfaces and was identified as Coccomyxa sp. KGU-D001 (Trebouxiophyceae). This study characterized its FA content and profile in a bath culture. The alga showed a constant specific growth rate (0.26 day^?1) ranging in light intensity from 20 to 80 μmol photons m^?2 s^?1. The algal cells started to form oil bodies in the early stationary phase of growth, and oil bodies occupied most of the cells during the late stationary phase when the cells accumulated 27 % total fatty acids (TFA). The process of lipid body formation accumulating large amounts of triacylglycerols (TAG) appeared to be very unusual in response to stress conditions persisting for a relatively long culture time (50 days). This study could indicate that aerial microalgae will be a candidate for biodiesel production when a new cultivation method is developed using extreme stresses such as nutritional deficiency and/or desiccation.     

Effect of Various Colors of Light-Emitting Diodes (LEDs) on the Biomass Composition of Arthrospira platensis Cultivated in Semi-continuous Mode

In the present study, semi-continuous cultivation of Arthrospira platensis using various colors of light-emitting diodes (LEDs) as artificial lighting was performed in order to study their effects on the biomass composition of A. platensis. The lowest biomass productivity was obtained with blue LED (4.68 mg l^?1 day^?1), while the highest was obtained with pink and red LEDs (30.89 and 30.69 mg l^?1 day^?1, respectively). All biomass compound contents were affected by the different colors studied, except that of total carotenoids. The lowest phycocyanin content was observed in pink LED (8.2 %) while the maximum in blue LED (17.6?±?2.4 %). Chlorophyll content was lowest in red LED (1.04 %) and highest in blue LED (1.42 %). The highest protein content was obtained with white and green LEDs (50.1 and 49.8 %, respectively), while the lowest was obtained with blue LED (42.1 %). Carbohydrate content was contrarily affected as that of proteins. The highest carbohydrate content was obtained in blue LED (11.3 %) and the lowest under white and pink LEDs (8.8 and 8.8 %, respectively). Lipid content seems to follow the same trend as that of carbohydrates; the highest lipid content was obtained in blue LED (6.0 %), and the lowest was obtained under pink LED (3.8 %).     

CO<Subscript>2</Subscript> Biofixation and Growth Kinetics of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Nannochloropsis gaditana</Emphasis>

CO₂ biofixation was investigated using tubular bioreactors (15 and 1.5 l) either in the presence of green algae Chlorella vulgaris or Nannochloropsis gaditana. The cultivation was carried out in the following conditions: temperature of 25 °C, inlet-CO₂ of 4 and 8 vol%, and artificial light enhancing photosynthesis. Higher biofixation were observed in 8 vol% CO₂ concentration for both microalgae cultures than in 4 vol%. Characteristic process parameters such as productivity, CO₂ fixation, and kinetic rate coefficient were determined and discussed. Simplified and advanced methods for determination of CO₂ fixation were compared. In a simplified method, it is assumed that 1 kg of produced biomass equals 1.88 kg recycled CO₂. Advance method is based on empirical results of the present study (formula with carbon content in biomass). It was observed that application of the simplified method can generate large errors, especially if the biomass contains a relatively low amount of carbon. N. gaditana is the recommended species for CO₂ removal due to a high biofixation rate—more than 1.7 g/l/day. On day 10 of cultivation, the cell concentration was more than 1.7?×?10⁷ cells/ml. In the case of C. vulgaris, the maximal biofixation rate and cell concentration did not exceed 1.4 g/l/day and 1.3?×?10⁷ cells/ml, respectively.     

<Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> application as a prospective approach for biosynthesis of pyruvic acid from glycerol

With the problems related to chemical methods of pyruvic acid (PA) synthesis, a fast-growing interest has been observed in research aiming at reducing the production cost of PA by applying biotechnological methods. This study aimed to investigate the potential applicability of Yarrowia lipolytica Wratislavia 1.31 yeast strain for valorisation of pure and crude glycerol through the production of industrially desired PA. Conditions required for the effective PA biosynthesis, i.e., pH value, thiamine concentration, agitation, and substrate concentration, were examined in batch and fed-batch cultivation modes. The efficient production of PA occurred under the limitation of thiamine (1 µg L^?1) and was stimulated by moderate pH (4.5) and agitation (800 rev min^?1) of the culture. Under optimal conditions, Y. lipolytica Wratislavia 1.31 was able to produce 85.2 g L^?1 of PA with volumetric productivity of 0.90 g L^?1 h^?1. The yield of PA biosynthesis reached a high level of 1.03 g g^?1. Obtained results confirmed the aptitude of Y. lipolytica yeast to produce high amounts of PA from simple glycerol-containing media. Presented process was very promising and might be considered as an attractive alternative for currently used chemical methods of PA synthesis.     

Low-Cost Production of Green Microalga Botryococcus braunii Biomass with High Lipid Content Through Mixotrophic and Photoautotrophic Cultivation

Botryococcus braunii is a microalga that is regarded as a potential source of renewable fuel because of its ability to produce large amounts of lipid that can be converted into biodiesel. Agro-industrial by-products and wastes are of great interest as cultivation medium for microorganisms because of their low cost, renewable nature, and abundance. In this study, two strategies for low-cost production of B. braunii biomass with high lipid content were performed: (i) the mixotrophic cultivation using molasses, a cheap by-product from the sugar cane plant as a carbon source, and (ii) the photoautotrophic cultivation using nitrate-rich wastewater supplemented with CO₂ as a carbon source. The mixotrophic cultivation added with 15 g L^?1 molasses produced a high amount of biomass of 3.05 g L^?1 with a high lipid content of 36.9 %. The photoautotrophic cultivation in nitrate-rich wastewater supplemented with 2.0 % CO₂ produced a biomass of 2.26 g L^?1 and a lipid content of 30.3 %. The benefits of this photoautotrophic cultivation are that this cultivation would help to reduce accumulation of atmospheric carbon dioxide and more than 90 % of the nitrate could be removed from the wastewater. When this cultivation was scaled up in a stirred tank photobioreactor and run with semi-continuous cultivation regime, the highest microalgal biomass of 5.16 g L^?1 with a comparable lipid content of 32.2 % was achieved. These two strategies could be promising ways for producing cheap lipid-rich microalgal biomass that can be used as biofuel feedstocks and animal feeds.     

Cultivation of Scenedesmus obliquus in Photobioreactors: Effects of Light Intensities and Light–Dark Cycles on Growth,Productivity, and Biochemical Composition

One of the main parameters influencing microalgae production is light, which provides energy to support metabolism but, if present in excess, can lead to oxidative stress and growth inhibition. In this work, the influence of illumination on Scenedesmus obliquus growth was assessed by cultivating cells at different light intensities in a flat plate photobioreactor. S. obliquus showed a maximum growth rate at 150 μmol photons m^?2 s^?1. Below this value, light was limiting for growth, while with more intense illumination photosaturation effects were observed, although cells still showed the ability to duplicate. Looking at the biochemical composition, light affected the pigment contents only while carbohydrate, lipid, and protein contents remained stable. By considering that in industrial photobioreactors microalgae cells are subjected to light–dark cycles due to mixing, algae were also grown under pulsed illumination (5, 10, and 15 Hz). Interestingly, the ability to exploit pulsed light with good efficiency required a pre-acclimation to the same conditions, suggesting the presence of a biological response to these conditions.     

Recycled de-Oiled Algal Biomass Extract as a Feedstock for Boosting Biodiesel Production from <Emphasis Type="Italic">Chlorella minutissima</Emphasis>

The investigation for the first time assesses the efficacy of recycled de-oiled algal biomass extract (DABE) as a cultivation media to boost lipid productivity in Chlorella minutissima and its comparison with Bold’s basal media (BBM) used as control. Presence of organic carbon (3.8 ± 0.8 g/l) in recycled DABE resulted in rapid growth with twofold increase in biomass productivity as compared to BBM. These cells expressed four folds higher lipid productivity (126 ± 5.54 mg/l/d) as compared to BBM. Cells cultivated in recycled DABE showed large sized lipid droplets accumulating 54.12 % of lipid content. Decrement in carbohydrate (17.76 %) and protein content (28.12 %) with loss of photosynthetic pigments compared to BBM grown cells were also recorded. The fatty acid profiles of cells cultivated in recycled DABE revealed the dominance of C16:0 (39.66 %), C18:1 (29.41 %) and C18:0 (15.82 %), respectively. This model is self-sustained and aims at neutralizing excessive feedstock consumption by exploiting recycled de-oiled algal biomass for cultivation of microalgae, making the process cost effective.     

Effects of pH and Carbon Source on <Emphasis Type="Italic">Synechococcus</Emphasis> PCC 7002 Cultivation: Biomass and Carbohydrate Production with Different Strategies for pH Control

Synechococcus PCC 7002 is an interesting species in view of industrial production of carbohydrates. The cultivation performances of this species are strongly affected by the pH of the medium, which also influences the carbohydrate accumulation. In this work, different methods of pH control were analyzed, in order to obtain a higher production of both Synechococcus biomass and carbohydrates. To better understand the influence of pH on growth and carbohydrate productivity, manual and automatic pH regulation in CO₂ and bicarbonate system were applied. The pH value of 8.5 resulted the best to achieve both of these goals. From an industrial point of view, an alternative way to maintain the pH practically constant during the entire period of cultivation is the exploitation of the bicarbonate-CO₂ buffer system, with the double aim to maintain the pH in the viability range and also to provide the amount of carbon required by growth. In this condition, a high concentration of biomass (6 g L^?1) and carbohydrate content (around 60 %) were obtained, which are promising in view of a potential use for bioethanol production. The chemical equilibrium of C-N-P species was also evaluated by applying the ionic balance equations, and a relation between the sodium bicarbonate added in the medium and the equilibrium value of pH was discussed.     

A New Temperature Control Shifting Strategy for Enhanced Triterpene Production by <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> G0119 Based on Submerged Liquid Fermentation

Temperature control is a very important factor on triterpene productivity in submerged liquid fermentation. Temperature effects from 23 to 32 °C on triterpene production by Ganoderma lucidum G0119 were investigated in 6-L stirred fermentor. Logistic and Luedeking-Piret equations were used to estimate the mycelial growth and triterpene production kinetics by regression analysis. On that basis, a temperature-shifting fermentation control strategy was established. From 0 to 61 h, culturing was performed at 32 °C to get high specific mycelial growth rate. Between 62 and 127 h, the temperature was decreased stepwise from 31 to 30 °C to maintain high triterpene productivity. After 128 h, temperature was maintained at 29 °C to minimize triterpene production inhibition and sustain high productivity. Elevated triterpene level (0.269 g L^?1), yield (0.0101 g g^?1), and productivity (0.00207 g (L h)^?1) were achieved representing 27.32, 13.94, and 37.11 % higher than submerged liquid fermentation at constant temperature of 29 °C, respectively, feasible for the industrial scale.     

Growth Parameters,Photosynthetic Performance,and Biochemical Characterization of Newly Isolated Green Microalgae in Response to Culture Condition Variations

This work aimed to characterize two native microalgal strains newly isolated from South Mediterranean areas and identified as Chlorella sorokiniana ES3 and Neochloris sp. AM2. The growth properties and biochemical composition of these microalgae were evaluated in different culture media (Algal, BG-11, f/2, and Conway). Among the tested media, nitrate- and phosphate-rich Algal medium provided the maximum biomass productivities (85.5 and 111.5 mg l^?1 day^?1 for C. sorokiniana and Neochloris sp., respectively), while the nitrate- and phosphate-deficient f/2 medium resulted in the highest lipid productivities (24.1 and 35.8 mg l^?1 day^?1 for C. sorokiniana and Neochloris sp., respectively). The physiological state of both microalgae was investigated under different light and temperature levels using the pulse amplitude-modulated fluorometry. The better photosynthetic efficiency of C. sorokiniana was obtained at 23 °C with a light saturation of 156 μE m^?2 s^?1, while that of Neochloris sp. was achieved at 15 °C with a light saturation of 151 μE m^?2 s^?1. The analysis of fatty acid profile and biodiesel parameters revealed that C. sorokiniana, cultivated in Algal and f/2 media, can be considered as a suitable candidate for high-quality biodiesel production.     

Single-cell Protein and Xylitol Production by a Novel Yeast Strain <Emphasis Type="Italic">Candida intermedia</Emphasis> FL023 from Lignocellulosic Hydrolysates and Xylose

Yeasts are good candidates to utilize the hydrolysates of lignocellulose, the most abundant bioresource, for bioproducts. This study aimed to evaluate the efficiencies of single-cell protein (SCP) and xylitol production by a novel yeast strain, Candida intermedia FL023, from lignocellulosic hydrolysates and xylose. This strain efficiently assimilated hexose, pentose, and cellubiose for cell mass production with the crude protein content of 484.2 g kg^?1 dry cell mass. SCP was produced by strain FL023 using corncob hydrolysate and urea as the carbon and nitrogen sources with the dry cell mass productivity 0.86 g L^?1 h^?1 and the yield of 0.40 g g^?1 sugar. SCP was also produced using NaOH-pretreated Miscanthus sinensis straw and corn steep liquor as the carbon and nitrogen sources through simultaneous saccharification and fermentation with the dry cell productivity of 0.23 g L^?1 h^?1 and yield of 0.17 g g^?1 straw. C. intermedia FL023 was tolerant to 0.5 g L^?1 furfural, acetic acid, and syringaldehyde in xylitol fermentation and produced 45.7 g L^?1 xylitol from xylose with the productivity of 0.38 g L^?1 h^?1 and the yield of 0.57 g g^?1 xylose. This study provides feasible methods for feed and food additive production from the abundant lignocellulosic bioresources.     

Realtime Monitoring of Xylitol Fermentation by Micro-Raman Spectroscopy

The fermentation of xylitol is a promising alternative to conventional chemical processes. Micro-Raman spectroscopy was used to monitor the process involving Candida tropicalis, including the medium and yeast cells during xylitol fermentation. The spectra of the fermentation medium showed that the characteristic xylitol peak at 866 cm^?1 was enhanced from 18 h and that the characteristic xylose peak at 901 cm^?1 gradually diminished as the reaction progressed. The characteristic ethanol peak at 880 cm^?1 indicated the production of by-products. Intracellular biological macromolecules, such as nucleic acids, proteins, lipids, and carbohydrates, were identified in the spectra of yeast cells. The intensity of nucleic acids at 783 cm^?1 reached the highest value after 3 h. The xylose band at 901 cm^?1 and the peaks in the carbohydrate region reached a maximum in the logarithmic phase, indicating the carbohydrate metabolism was the most active. The amide I band located at 1658 cm^?1 indicated the major secondary structure of proteins was α-helix; its intensity gradually reduced during the fermentation. The 853 cm^?1 band due to buried tyrosine was predominant at 21 h. In addition, the 1275 cm^?1 band corresponded to the presence of a random coil only at 27 h. These results provided a perspective to understand fermentation and verified the applicability of Raman spectroscopy in xylitol fermentation.     

Ethanol Production from Starch Hydrolyzates using <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> and Glucoamylase Entrapped in Polyvinylalcohol Hydrogel

The glucoamylase from Aspergillus niger, immobilized into poly(vinylalcohol) hydrogel lens-shaped capsules LentiKats®, was used for simultaneous saccharification and fermentation (SSF) with Zymomonas mobilis in free form. This system was stable in both the repeated batch and continuous mode of SSF. The microorganism was found to adsorb on the capsules with immobilized enzyme. This increased the ethanol productivity of the repeated batch system with 5% w/v of immobilized glucoamylase almost 2.1 times (7.2 g l^?1 h^?1) compared to free enzyme–free microorganism system (3.5 g l^?1 h^?1). The continuous SSF with the immobilized glucoamylase (11.5% w/v) tested for 15 days had productivity 10 g l^?1 h^?1, which is comparable to continuous experiments on semi-defined glucose medium (10 g l^?1 h^?1). These two systems were stable in both glucoamylase activity and microorganism productivity.