Screening,Growth Medium Optimisation and Heterotrophic Cultivation of Microalgae for Biodiesel Production

This article presents a study on screening of microalgal strains from the Peking University Algae Collection and heterotrophic cultivation for biodiesel production of a selected microalgal strain. Among 89 strains, only five were capable of growing under heterotrophic conditions in liquid cultures and Chlorella sp. PKUAC 102 was found the best for the production of heterotrophic algal biodiesel. Composition of the growth medium was optimised using response surface methodology and optimised growth conditions were successfully used for cultivation of the strain in a fermentor. Conversion of algal lipids to fatty acid methyl esters (FAMEs) showed that the lipid profile of the heterotrophically cultivated Chlorella sp. PKUAC 102 contains fatty acids suitable for biodiesel production.     

Isolation,Identification and High-Throughput Screening of Neutral Lipid Producing Indigenous Microalgae from South African Aquatic Habitats

Exploring indigenous microalgae capable of producing significant amounts of neutral lipids through high-throughput screening is crucial for sustainable biodiesel production. In this study, 31 indigenous microalgal strains were isolated from diverse aquatic habitats in KwaZulu-Natal, South Africa. Eight superior lipid-producing strains were selected for further analysis, based on Nile red fluorescence microscopy screening. The microalgal isolates were identified to belong to the genera Chlorella, Neochloris and Chlamydomonas via morpho-taxonomic and molecular approach by 18S rRNA gene sequencing. Chlorella vulgaris PH2 had the highest specific growth rate (μ) and lowest doubling time of 0.24 day⁻¹ and 2.89 ± 0.05 day⁻¹, respectively. Chlorella vulgaris T4 had the highest biomass productivity of 35.71 ± 0.03 mg L⁻¹day⁻¹. Chlorella vulgaris PH2 had the highest lipid content of 34.28 ± 0.47 and 38 ± 9.2% (dcw) as determined by gravimetric analysis and the sulfo-phospho-vanillin (SPV) method, respectively. Chlorella vulgaris PH2 exhibited a high content of saturated fatty acids, while Chlorella sp. T4 exhibited a high total content of saturated and monounsaturated fatty acids with a low content of polyunsaturated fatty acids. The preponderance of neutral lipids suggests that Chlorella sp. T4 is a suitable candidate for biomass feedstock for biodiesel production.

     

Marine Diatom, Navicula sp. Strain JPCC DA0580 and Marine Green Alga, Chlorella sp. Strain NKG400014 as Potential Sources for Biodiesel Production

Marine diatom, strain JPCC DA0580, and marine green microalga strain NKG400014 were selected as high neutral lipid-producers from marine microalgal culture collection toward biodiesel production. These strains were tentatively identified as Navicula sp. and Chlorella sp., respectively, by 18S rDNA analysis. Growth and lipid accumulation conditions of both strains were analyzed by changing nutrient concentrations in growth media and initial illuminance intensity. The highest productivity of fatty acid methyl ester (FAME) reached to 154 mg/L/week for NKG400014 and 185 mg/L/week for JPCC DA0580. Gas chromatography/mass spectrometry analysis indicates that FAME fraction from NKG400014 mainly contained 9-12-15-octadecatrienoate (C18:3) and that from JPCC DA0580 mainly contained methyl palmitate (C16:0) and methyl palmitoleate (C16:1). Furthermore, calorimetric analysis revealed that the energy content of strain was 4,233?±?55 kcal/kg (i.e., 15.9?±?0.2 MJ/kg) for NKG400014 and 6,423?±?139 kcal/mg (i.e., 26.9?±?0.6 MJ/kg) for JPCC DA0580, respectively. The value from JPCC DA0580 was equivalent to that of coal. The strains NKG400014 and JPCC DA0580 will become a promising resource that can grow as dominant species in the open ocean toward production of both liquid and solid biofuels.     

Isolation of thermo-stable and solvent-tolerant Bacillus sp. lipase for the production of biodiesel

This study presents the production of biodiesel from algae oil by transesterification using thermophilic microorganism. The microorganism used in this study was isolated from the soil sample obtained near the furnace. The organism was identified as Bacillus sp., and the lipase obtained was purified by ammonium sulfate precipitation and ion exchange chromatography leading to 8.6-fold purification and 13% recovery. Molecular weight of the enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was found to be 45 kDa. The effect of pH, temperature, and solvent addition on lipase activity was investigated. The enzyme showed maximum activity at 55 °C and at pH 7 and was also found to be highly active in the presence of organic solvents such as hexane and t-butanol. The isolated lipase was successfully used for the production of biodiesel. The transesterification activity of the isolated lipase showed 76% of fatty acid methyl esters yield in 40 h, which indicated that this enzyme can be used as a potential biocatalyst for the biodiesel production.     

Industrial Waste Utilization for Low-Cost Production of Raw Material Oil Through Microbial Fermentation

In view of ever-growing demand of biodiesel, there is an urgent need to look for inexpensive and promising renewable raw material oils for its production. In this context, the aim of this study was to evaluate the potential use of industrial wastes for low-cost production of oils through microbial fermentation. Among the strains tested, Yarrowia lipolytica grew best and produced highest lipid when grown on decanter effluent from palm oil mill. When crude glycerol by-product from a biodiesel plant was added into the effluent as a co-substrate, Y. lipolytica produced a higher biomass of 3.21 g/L and a higher amount of lipid of 2.21 g/L which was 68 % of the dry weight. The scale up and process improvement in a 5-L bioreactor increased the biomass and lipid up to 5.53 and 2.81 g/L, respectively. A semi-continuous mode of operation was an effective mode for biomass enhancement while a fed-batch mode was effective for lipid enhancement. These yeast lipids have potential to be used as biodiesel feedstocks because of their similar fatty acid composition to that of plant oil.     

Triangle of AKT2, miRNA,and Tumorigenesis in Different Cancers

Producing biodiesel from microalgae grown in wastewater is environment-friendly and cost-effective. The present study investigated the algae found in wastewater of a local dairy farm for their potential as biodiesel feedstocks. Thirteen native algal strains were isolated. On the basis of morphology and 16S/18S rRNA gene sequences, one strain was identified to be a member of cyanobacteria, while other 12 strains belong to green algae. After screening, two Scenedesmus strains out of the 13 microalgae isolates demonstrated superiority in growth rate, lipid productivity, and sedimentation properties, and therefore were selected for further scale-up outdoor cultivation. Both Scenedesmus strains quickly adapted to the outdoor conditions, exhibiting reasonably good growth and strong anti-contamination capabilities. In flat-plate photobioreactors (PBRs), algal cells accumulated predominantly neutral lipids that accounted for over 60% of total lipids with almost 70% being triacylglycerol. In addition, Scenedesmus obliquus had a high content of monounsaturated fatty acids, of which the amount of oleic acid (C18:1) was up to 27.11%. Based on these findings, the dairy farm wastewater-isolated Scenedesmus strains represent promising sources of low-cost, high-quality oil for biofuel production.     

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.     

Metabolism of methylated arsenic compounds by arsenic-resistant bacteria (Klebsiella oxytoca and Xanthomonas sp.)

Two bacteria exhibiting resistance to toxic arsenic were isolated. These had been contaminated with arsenic in a Chlorella sp. culture medium containing arsenic. The two bacteria were identified as Klebsiella oxytoca and Xanthomonas sp., and grew well in a peptone medium at neutral pH at 30°C, reaching the stationary phase in ca 100h and 70h, respectively. The growth of the bacteria was not affected by arsenic(V) concentrations in the medium as high as 1000mg dm^?3. The bacteria bioaccumulated arsenic, a part of the arsenic being methylated. The bioaccumulation exhibited its peak around the turing point from the log phase to the stationary phase. The relative content of methylated arsenic in the excrement was greater than that in the bacterial cells. Adaptation treatment of inorganic arsenic caused an increase in the bioaccumulation of inorganic arsenic by K. oxytoca. Such a situation was not observed in the case of Xanthomonas sp. The bacteria also bioaccumulated methylated arsenic compounds, and demethylation of these species was observed. When the bacteria were killed by ethanol, arsenic was not taken up by the cells.     

Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by <Emphasis Type="Italic">in situ</Emphasis> transesterification

In the context of algal biofuels, lipids, or better aliphatic chains of the fatty acids, are perhaps the most important constituents of algal biomass. Accurate quantification of lipids and their respective fuel yield is crucial for comparison of algal strains and growth conditions and for process monitoring. As an alternative to traditional solvent-based lipid extraction procedures, we have developed a robust whole-biomass in situ transesterification procedure for quantification of algal lipids (as fatty acid methyl esters, FAMEs) that (a) can be carried out on a small scale (using 4–7 mg of biomass), (b) is applicable to a range of different species, (c) consists of a single-step reaction, (d) is robust over a range of different temperature and time combinations, and (e) tolerant to at least 50% water in the biomass. Unlike gravimetric lipid quantification, which can over- or underestimate the lipid content, whole biomass transesterification reflects the true potential fuel yield of algal biomass. We report here on the comparison of the yield of FAMEs by using different catalysts and catalyst combinations, with the acid catalyst HCl providing a consistently high level of conversion of fatty acids with a precision of 1.9% relative standard deviation. We investigate the influence of reaction time, temperature, and biomass water content on the measured FAME content and profile for 4 different samples of algae (replete and deplete Chlorella vulgaris, replete Phaeodactylum tricornutum, and replete Nannochloropsis sp.). We conclude by demonstrating a full mass balance closure of all fatty acids around a traditional lipid extraction process.     

Cultivating <Emphasis Type="BoldItalic">Chlorella</Emphasis> sp. in a Pilot-Scale Photobioreactor Using Centrate Wastewater for Microalgae Biomass Production and Wastewater Nutrient Removal

This study is concerned with a novel mass microalgae production system which, for the first time, uses “centrate”, a concentrated wastewater stream, to produce microalgal biomass for energy production. Centrate contains a high level of nutrients that support algal growth. The objective of this study was to investigate the growth characteristics of a locally isolated microalgae strain Chlorella sp. in centrate and its ability to remove nutrients from centrate. A pilot-scale photobioreactor (PBR) was constructed at a local wastewater treatment plant. The system was tested under different harvesting rates and exogenous CO₂ levels with the local strain of Chlorella sp. Under low light conditions (25 μmol·m^-2s^-1) the system can produce 34.6 and 17.7 g·m^-2day^-1 biomass in terms of total suspended solids and volatile suspended solids, respectively. At a one fourth harvesting rate, reduction of chemical oxygen demand, total Kjeldahl nitrogen, and soluble total phosphorus were 70%, 61%, and 61%, respectively. The addition of CO₂ to the system did not exhibit a positive effect on biomass productivity or nutrient removal in centrate which is an organic carbon rich medium. The unique PBR system is highly scalable and provides a great opportunity for biomass production coupled with wastewater treatment.     

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.     

Biopolymer Synthesized by Strains of Xanthomonas sp Isolate from Brazil Using Biodiesel-Waste

The future supplies and usage of glycerol are expected to increase as biodiesel plants increase production, and the output will greatly outpace demand. Biodiesel production has already had a significant impact on the price of refined glycerol. A major concern of glycerol producers is the reduced price of glycerol resulting from the increased production of biodiesel. Some alternative uses for this glycerol that have been investigated are substrates for fermentation process or the production of biosurfactants, fatty acids, biopolymers, and others products. This work had as objective to evaluate two strains of Xanthomonas sp isolate from Brazil for xanthan gum in orbital agitator, analizing the apparent viscosity of aqueous solutions and selecting viscosity. The experiments of xanthan gum production were realized in orbital agitator with 120 rpm agitation, for cells production, and 180rpm, for biopolymer production, under a 28 °C temperature. The rheology of the fermentation broth was analyzed by apparent viscosity and the polymer was recovered with ethanol (1:3, v/v). After its recovery, the productivity evaluation was performed. The productivity were 0.157 and 0.363 gL⁻¹ for C1 and 0.186 and 0.363 gL⁻¹ for C9 to media glycerol or glycerol and sucrose, respectively. The viscosity analysis was performed for aqueous solutions 3%, at 25 °C, the best apparent viscosity was obtained using gum synthesized with glycerol and sucrose (50:50) at 25 °C, 143 mPa.s⁻¹ from Xanthomonas sp C1.     

Thermal and rheological behavior of diesel and methanol biodiesel blends

The most feasible alternative among fuels derived from biomass seems to be the biodiesel, having the required characteristics for a total or partial substitution of diesel oil. Therefore, the aim of this work is to evaluate the thermal and rheological behavior of the blends of diesel with the methanol biodiesel obtained from soybean oil, using B5, B15 and B25 blends. All thermogravimetric curves exhibited one overlapping mass loss step in the 35–280°C temperature range at air atmosphere and one step between 37–265°C in nitrogen. The rheological study showed a Newtonian behavior (n=1) for all blends.     

Evaluation of the effects of crude glycerol on the production and properties of novel polyhydroxyalkanoate copolymers containing high 11‐hydroxyoctadecanoate by Cupriavidus necator IPT 029 and Bacillus megaterium IPT 429

Crude glycerol (CG), a by‐product from biodiesel production, is a carbon source with potential as feedstock for polyhydroxyalkanoate (PHA) production. PHAs are biological macromolecules synthesized by microorganisms as intracellular carbon and energy storage granules. PHA production and its properties were investigated using Cupriavidus necator IPT 029 and Bacillus megaterium IPT 429 cultivated with CGs from different origins. The highest PHA extraction percentage (71.56% [w/v]) occurred when C. necator IPT 029 metabolized CG 3 (from the processing of biodiesel from castor bean oil). The gas chromatography–mass spectrometry analyses revealed novel PHA constituents as building blocks of medium (3‐hydroxytetradecanoate) and long (11‐hydroxyoctadecanoate) chains. Molar mass distribution revealed range of 121–6900 kDa. The initial degradation temperature ranged from 181.83 to 287.50°C and the crystallinity ranged from 35.30 to 66.70%. The results obtained indicate that C. necator IPT 029 from CG 3 could produce copolymers with industrially applicable thermophysical properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Utilization of waste seashells and Camelina sativa oil for biodiesel synthesis

The seafood industry produces over 100 million pounds of seashell waste every year. With landfill space diminishing quickly, ways to recycle waste materials are becoming more sought for. Herein, we utilized waste mussel, clam and oyster shells as heterogeneous catalysts for the transesterification of Camelina sativa oil as a feedstock into biodiesel. Camelina sativa oil provides a reliable solution for biodiesel production because it has high tolerance of cold weather, drought, low-fertility soils and contains approximately 40% oil content. The catalysts from waste seashells were characterized by X-ray powder diffraction and Fourier transform infrared (FTIR) spectroscopy. High biodiesel yields were achieved at a 12:1 (alcohol:oil) molar ratio with 1?wt.% waste seashell catalysts in 2?h at 65°C. Biodiesel was analyzed by ¹H NMR and FTIR spectroscopy and the fuel properties of the biodiesel produced from Camelina sativa oil and waste seashells were compared with American Society for Testing and Materials standards.     

Exploratory Analysis for Elemental Characterization of Biomass Residues From Biodiesel Production by Inductively Coupled Plasma Optical Emission Spectrometry

Edible and non-edible seeds with high oil content are used as the main raw material for biodiesel production. The residual biomass (press cake or oil cake) can be used as animal feed, to aggregate value to the biodiesel production cycle. In this work, a microwave-assisted-digestion procedure for elementary determination by inductively coupled plasma optical emission spectrometry (ICP OES) using dilute nitric acid was developed. Inter-laboratorial reference material was used to check the accuracy. A full factorial design in addition to exploratory analysis through principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to classify the samples: press cakes from castor (Ricinus communis), jatropha (Jatropha curcas), radish (Raphanus sativus), sunflower (Heliantus annus), and coffee (Coffe arabica), according to their similarities.     

Spectroscopic determination of U(VI) species sorbed by the Chlorella (Chlorella pyrenoidosa) fresh water algae

The green algae Chlorella (Chlorella pyrenoidosa) have the ability to bind high amounts of uranium(VI) in the pH range from 3 to 6. At pH 3 up to 20 % of the uranium is bound by the algal cells whereas the uranium removal by algal cell is almost complete at pH 5 and 6 in the concentration range of 4 × 10^?4 to 1.6 × 10^?3 M. Sorption capacities are in the range of 300–350 mg g^?1 and 250–280 mg g^?1 for fresh water and seawater respectively. Concentration of uranium was measured by inductively coupled plasma optical emission spectroscopy (ICP-OES) by using two different emission spectral lines at 409.014 and 424.167 nm. Environmental scanning electron microscopy (ESEM) complimented with energy dispersive X-ray (EDX) is used to characterize the binding sites of uranyl species on algal cell in the selected pH range. The micrographs show a regular distribution of U(VI) on the cell surface. Attenuated total reflectance-fourier transform infrared (ATRFTIR) spectrum of Chlorella indicates that the binding of U(VI) either to phosphodiesters (P–O–aryl/P–O–alkyl), and combination of amine, secondary amine and imine = NH respectively. These sites in Chlorella groups are mainly responsible for the removal and binding of U(VI) by formation of organic and/or inorganic uranyl phosphates.     

The Utilization of Post-chlorinated Municipal Domestic Wastewater for Biomass and Lipid Production by <Emphasis Type="Italic">Chlorella</Emphasis> spp. Under Batch Conditions

South Africa has a rich microalgal biodiversity which has the potential to be used for renewable bio-fuel production in the region. Bioprospecting for oleaginous microalgae in KwaZulu Natal Province, South Africa, resulted in the establishment of a microalgal culture collection system for alternative energy research in the country. A potential hyper-lipid-producing Chlorella spp. strain was isolated, purified, and cultured in supplemented post-chlorinated wastewater for biomass and lipid production at the laboratory scale under batch mode. The microalgal strain was cultivated in different strengths of BG-11 media supplemented with wastewater from a local municipal domestic wastewater treatment plant. The Chlorella spp. was grown using ambient dissolved carbon dioxide in shake flasks under photosynthetically active radiation (±120 μmolm⁻²s⁻¹). Microalgal biomass and lipid productivity were monitored at 24-h intervals in the batch mode. The microalgal biomass was analyzed by direct light microscopy and indirectly by spectrophotometry at 600 nm, and the lipids were extracted and quantified. The growth rate of the Chlorella spp. was enhanced in post-chlorinated wastewater supplemented with 5 mM NaNO₃ with maximal biomass productivity. A dramatic increase in lipid yield was achieved with the post-chlorinated wastewater supplemented with 25 mM NaNO₃. Low dosages of free chlorine were found to enhance microalgal growth. These findings serve as a basis for further scale-up trials using municipal wastewater as a medium for microalgal biomass and lipid production.     

Bioconversion of Corncob Acid Hydrolysate into Microbial Oil by the Oleaginous Yeast Lipomyces starkeyi

For the first time, corncob acid hydrolysate was used for microbial oil production by the oleaginous yeast Lipomyces starkeyi. After hydrolysis by dilute sulfuric acid, corncob could turn into an acid hydrolysate with a sugar concentration of about 42.3 g/L. Detoxified by overliming and absorption with activated carbon, the corncob hydrolysate could be used by L. starkeyi efficiently that a total biomass of 17.2 g/L with a lipid content of 47.0 % (corresponding to a lipid yield of 8.1 g/L) and a lipid coefficient of 20.9 could be obtained after cultivation on the corncob hydrolysate for 8 days. Therefore, L. starkeyi is a promising strain for microbial oil production from lignocellulosic biomass. Glucose and xylose were used by L. starkeyi simultaneously during lipid fermentation while arabinose could not be utilized by it. Besides, the lipid composition of L. starkeyi was similar to that of vegetable oils; thus, it is a promising feedstock for biodiesel production.     

Biotechnological Utilization of Biodiesel-Derived Glycerol for the Production of Ribonucleotides and Microbial Biomass

Ten yeast strains were evaluated concerning their capabilities to assimilate biodiesel-derived glycerol in batch cultivation. The influence of glycerol concentration, temperature, pH and yeast extract concentration on biomass production was studied for the yeast selected. Further, the effect of agitation on glycerol utilization by the yeast Hansenula anomala was also studied. The yeast H. anomala CCT 2648 showed the highest biomass yield (0.30?g?g^?1) and productivity (0.19?g?L^?1?h^?1). Citric acid, succinic acid, acetic acid and ethanol were found as the main metabolites produced. The increase of yeast extract concentration from 1 to 3?g?L^?1 resulted in high biomass production. The highest biomass concentration (21?g?L^?1), yield (0.45?g?g^?1) and productivity (0.31?g?L^?1?h^?1), as well as ribonucleotide production (13.13?mg?g^?1), were observed at 700?rpm and 0.5?vvm. These results demonstrated that glycerol from biodiesel production process showed to be a feasible substrate for producing biomass and ribonucleotides by yeast species.