Heterotrophic Culture of <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> in Various Nitrogen Sources for Lipid Production

The influences of urea, yeast extract, and nitrate as the nitrogen source on heterotrophic growth of four strains of Chlorella protothecoides were investigated in 9-day feed-batch cultures. Biomass dry weight concentration (DWC) and lipid yield (LY) of the four strains in all media were compared. The highest LY in 9 days was 654 mg/L/day by UTEX 255 in 2.4 g/L KNO₃ medium with a biomass DWC of 11.7 g/L and lipid content of 50.5%. Using green autotrophic seeds instead of yellow heterotrophic seeds improved the biomass DWC (13.1 vs. 11.7 g/L), LY (850 vs. 654 mg/L/day), and lipid to glucose consumption ratio (0.607 vs. 0.162). Moreover, 17.0 g/L DWC and 489 mg/L/day LY were obtained from the sequentially mixed-nitrogen medium, and the lipid to glucose consumption ratio was improved to 0.197 from 0.162 in 2.4 g/L nitrate medium and from 0.108 in 4.2 g/L yeast extract medium in the first batch.     

Influence of Exogenous CO2 on Biomass and Lipid Accumulation of Microalgae Auxenochlorella protothecoides Cultivated in Concentrated Municipal Wastewater

The effects of exogenous CO₂ on the growth and lipid accumulation of a local screened facultative heterotrophic microalgae strain Auxenochlorella protothecoides (UMN280) as well as nutrient removal from concentrated municipal wastewater stream (centrate) were examined in this study. A 12-day batch experiment was conducted with CO₂ aeration at three levels, namely, 0%, 1%, and 5% (v/v) CO₂ mixed with air, under light intensity of 60???mol/(m² @@s). A two-stage growth pattern was observed. The first stage (first?Cfifth day) was dominated by heterotrophic growth in which organic carbon was the main carbon source. The second stage (6th?C12th day) was dominated by autotrophic growth in which exogenous CO₂ had a positive effect on algal biomass accumulation. The addition of 5% CO₂ was better than that of 1% CO₂ on the biomass and lipid production. The uptakes of nutrients were similar between injection and no injection of CO₂, except on phosphorus removal which was affected by the acidification of CO₂.     

Fermentation Characteristics of <Emphasis Type="Italic">Mortierella alpina</Emphasis> in Response to Different Nitrogen Sources

The fermentation characteristics of Mortierella alpina were investigated in response to various nitrogen sources. Influences on nitrogen source and glucose uptake rate, mycelial morphology of M. alpina, and pH of medium in relation to different nitrogen sources were discussed. Effects of different nitrogen sources on cell growth, fatty acid composition, arachidonic acid (ARA), and total lipid concentration were also evaluated. It revealed that the maximum nitrogen source uptake ratio was obtained when corn steep liquor was used as nitrogen source. When yeast extract was used as the sole nitrogen source, glucose was completely exhausted at the end of fermentation. The maximum dry cell weight obtained from medium with yeast extract as nitrogen source had the highest total lipid concentration. Sodium nitrate was the favorable nitrogen source for ARA accumulation, and the highest ARA percentage in total fatty acids was obtained, 35.9%. Urea was identified as the favorable nitrogen source for ARA production, the highest ARA concentration obtained from urea was 5.8 g/l. Compared with inorganic nitrogen sources, organic nitrogen compounds are favorable for both cell growth and total lipids accumulation.     

微藻高油脂化基因工程研究策略

石化能源危机与全球气候变化已成为人类的两大重要难题。生物柴油作为可替代普通柴油的环境友好且可再生的能源受到普遍关注。相比植物油和动物脂肪生产,藻类油脂产率高且容易培养,被认为是未来生物柴油发展的重要原料之一。通过转基因技术强化油脂代谢途径,提高富油微藻含油量,已经成为新的研究热点。已有植物研究表明,增强甘油酯酰基转移酶表达,可以提高Kennedy途径代谢中间体通量,从而增加甘油三酯(TAG)的积累。本文综述了微藻油脂代谢途径的国内外研究现状和提高油脂积累的代谢调控策略;详细阐述了基于植物油脂合成强化的成功经验,通过增强微藻Kennedy途径对提高TAG生物合成的重要作用;讨论了当前转基因微藻的遗传转化方法及其需要解决的关键性科学技术问题;分析了基因工程技术调控微藻脂类代谢途径生产高油脂的可能性,并对该研究的发展进行了展望。     

Metabolomic Study of Heterotrophically Grown Chlorella sp. Isolated from Wastewater in Northern Sweden

There are numerous strains of Chlorella with a corresponding variety of metabolic pathways. A strain we previously isolated from wastewater in northern Sweden can grow heterotrophically as well as autotrophically in light and has higher lipid contents under heterotrophic growth conditions. The aims of the present study were to characterize metabolic changes associated with the higher lipid contents in order to enhance our understanding of lipid production in microalgae and potentially identify new compounds with utility in sustainable development. Inter alia, the amino acids glutamine and lysine were 7-fold more abundant under heterotrophic conditions, the key metabolic intermediate alpha-ketoglutarate was more abundant under heterotrophic conditions with glucose, and maltose was more abundant under heterotrophic conditions with glycerol than under autotrophic conditions. The metabolite 3-hydroxy-butyric acid, the direct precursor of the biodegradable plastic PHB (poly-3-hydroxy-butyric acid), was also more abundant under heterotrophic conditions. Our metabolomic analysis has provided new insights into the alga’s lipid production pathways and identified metabolites with potential use in sustainable development, such as the production of renewable, biodegradable plastics, cosmetics, and nutraceuticals, with reduced pollution and improvements in both ecological and human health.     

Carbohydrate metabolite pathways and antibiotic production variations of a novel Streptomyces sp. M3004 depending on the concentrations of carbon sources

To determine the variations of growth, some key enzyme activities such as glucose kinase (GK), glucose-6-phosphate dehydrogenase (G6PDH), α-ketoglutarate dehydrogenase (KGDH), and isocitrate lyase (ICL) besides metabolite levels of pyruvate and antibiotic production of newly isolated Streptomyces sp. M3004 were grown in culture media which contain 10–20 g/l concentration with either glucose or glycerol as carbon source. Biomass and intracellular glucose and glycerol levels of Streptomyces sp. M3004 showed positive correlation with the concentration of these carbon sources, and these levels were higher in glucose compared with the glycerol-supplemented mediums. GK, G6PDH, and KGDH activities showed marked correlation with the concentration of both glucose and glycerol, and the activity levels were 4.14-, 1.47-, and 1.27-fold higher in glucose than glycerol. A key enzyme of the glyoxalate cycle, ICL activities decreased with increasing glucose concentrations from 10 to 20 g/l, but increased up to 15 g/l of glycerol. The positive correlations were also determined between intracellular glucose and glycerol levels besides pyruvate and protein variations with respect to concentrations of the carbon sources. Antibacterial activities of Streptomyces sp. M3004 reached maximum on the stationary phase, while it did not change significantly with respect to glucose and glycerol.     

Production of Oils from Acetic Acid by the Oleaginous Yeast Cryptococcus curvatus

The feasibility of the conversion of acetic acid, a metabolite commonly obtained during anaerobic fermentation processes, into oils using the yeast Cryptococcus curvatus was reported. This microorganism exhibited very slow growth rates on acetate as carbon source, which led to design a two-stage cultivation process. The first consisted of cell growth on glucose as carbon source until its complete exhaustion. The second step involved the use of acetate as carbon source under nitrogen limitation in order to induce lipid accumulation. A typical experiment performed in a bioreactor involved a preliminary yeast growth with a glucose initial concentration of 15?g/L glucose. Further additions of acetate and nitrogen source allowed a final lipid accumulation up to 50% (w/w). These promising results demonstrated the suitability of the technique proposed.     

Hexavalent Molybdenum Reduction to Molybdenum Blue by <Emphasis Type="Italic">S. Marcescens</Emphasis> Strain Dr. Y6

A molybdate-reducing bacterium has been locally isolated. The bacterium reduces molybdate or Mo⁶⁺ to molybdenum blue (molybdate oxidation states of between 5+ and 6+). Different carbon sources such as acetate, formate, glycerol, citric acid, lactose, fructose, glucose, mannitol, tartarate, maltose, sucrose, and starch were used at an initial concentration of 0.2% (w/v) in low phosphate media to study their effect on the molybdate reduction efficiency of bacterium. All of the carbon sources supported cellular growth, but only sucrose, maltose, glucose, and glycerol (in decreasing order) supported molybdate reduction after 24 h of incubation. Optimum concentration of sucrose for molybdate reduction is 1.0% (w/v) after 24 h of static incubation. Ammonium sulfate, ammonium chloride, valine, OH-proline, glutamic acid, and alanine (in the order of decreasing efficiency) supported molybdate reduction with ammonium sulfate giving the highest amount of molybdenum blue after 24 h of incubation at 0.3% (w/v). The optimum molybdate concentration that supports molybdate reduction is between 15 and 25 mM. Molybdate reduction is optimum at 35 °C. Phosphate at concentrations higher than 5 mM strongly inhibits molybdate reduction. The molybdenum blue produced from cellular reduction exhibits a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. The isolate was tentatively identified as Serratia marcescens Strain Dr.Y6 based on carbon utilization profiles using Biolog GN plates and partial 16s rDNA molecular phylogeny.     

Mixotrophic Cultivation of Microalgae for Biodiesel Production: Status and Prospects

Biodiesel from microalgae provides a promising alternative for biofuel production. Microalgae can be produced under three major cultivation modes, namely photoautotrophic cultivation, heterotrophic cultivation, and mixotrophic cultivation. Potentials and practices of biodiesel production from microalgae have been demonstrated mostly focusing on photoautotrophic cultivation; mixotrophic cultivation of microalgae for biodiesel production has rarely been reviewed. This paper summarizes the mechanisms and virtues of mixotrophic microalgae cultivation through comparison with other major cultivation modes. Influencing factors of microalgal biodiesel production under mixotrophic cultivation are presented, development of combining microalgal biodiesel production with wastewater treatment is especially reviewed, and bottlenecks and strategies for future commercial production are also identified.     

Production of Lipid and Fatty Acids during Growth of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> on Hydrocarbon Substrates

An Aspergillus terreus, isolated from oil contaminated soil, could degrade a wide range of petroleum hydrocarbons including the immediate oxidation products of hydrocarbons, like alkanols and alkanals. Among all the linear chain carbon substrates, highest growth of 39.1 ± 3.8 g l⁻¹ (wet weight) was observed when n-hexadecane was used as the sole source of carbon. The growth of the fungus on this highly hydrophobic substrate was associated with the morphological change of the hyphae and increase production of lipid in the cells. The lipid production in the hydrocarbon (n-hexadecane) grown cells was sevenfold higher than the corresponding glucose grown cells. The fatty acid profile of the lipid content formed in the hydrocarbon grown cells was significantly different from the glucose grown cells and was composed of fatty acids with chain length C₁₄ to C₃₃ as revealed from the liquid chromatography electrospray ionization mass spectrometry analyses. Among the ranges, the fatty acids with chain lengths C₁₄ to C₁₈ were predominant in the profile. Considering the fatty acid profile and the high level of lipid production, this A. terreus mediated production of lipid is envisaged to have potential application in the oleochemical industries including the production of biodiesel.     

Lignin peroxidase production by Streptomyces viridosporus T7A

Lignin peroxidase (LiP) production cost should be reduced to justify its use in the control of environmental pollution. In this work, we studied the enzyme production by Streptomyces viridosporus T7A using glucose or corn oil as a carbon source having 0.65% yeast extract as a nitrogen source. Enzyme activity, observed using either 0.65% glucose or corn oil at 0.1, 0.5, and 1.0% concentration, was 300, 150, 300, and 200 U/L, respectively. Although higher enzyme activity was obtained in both media containing 0.65% glucose and 0.5% corn oil, the use of corn oil resulted in a better LiP stability. When combined carbon sources were used, higher values of enzyme activity (360, 350, and 225 U/L) were observed in media with 0.65% glucose and supplemented with 0.1, 0.5, and 1.0% corn oil, respectively. Although the presence of both glucose and 0.5% corn oil is favorable for LiP production, satisfactory results in terms of enzyme production and stability could be also observed using 0.5% corn oil as a sole carbon source, which may lead to reduced production costs of the LiP enzyme.     

Microbial production of polyhydroxyalkanoates by bacteria isolated from oil wastes

A Gram-positive coccus-shaped bacterium capable of synthesizing higher relative molecular weight (M _r), polyhydroxybutyrate (PHB) was isolated from sesame oil and identified as Staphylococcus epidermidis (by Microbial ID, Inc., Newark, NJ). The experiment was conducted by shake flask fermentation culture using media containing fructose. Cell growth up to a dry mass of 2.5 g/L and PHB accumulation up to 15.02% of cell dry wt was observed. Apart from using single carbohydrate as a sole carbon source, various industrial food wastes including sesame oil, ice cream, malt, and soya wastes were investigated as nutrients for S. epidermidis to reduce the cost of the carbon source. As a result, we found that by using malt wastes as nutrient for cell growth, PHB accumulation of S. epidermidis was much better than using other wastes as nutrient source. The final dried cell mass and PHB production using malt wastes were 1.76 g/L and 6.93% polymer/cells (grams/gram), and 3.5 g/L and 3.31% polymer/cells (grams/gram) in shake flask culture and in fermentor culture, respectively. The bacterial polymer was characterized by ¹H-nuclear magnetic resonance (NMR), ¹³C-NMR, Fourier transform infrared, and differential scanning calorimetry. The results show that with different industrial food wastes as carbon and energy sources, the same biopolymer (PHB) was obtained. However, the use of sesame oil as the carbon source resulted in the accumulation of PHB with a higher melting point than that produced from other food wastes as carbon sources by this organism under similar experimental conditions.     

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.     

Production of lipase by a newly isolated <Emphasis Type="Italic">Bacillus coagulans</Emphasis> under solid-state fermentation using melon wastes

Summary An extracellular lipase was produced by Bacillus coagulans by solid-state fermentation. Solid waste from melon was used as the basic nutrient source and was supplemented with olive oil. The highest lipase production (78,069 U/g) was achieved after 24h of cultivation with 1% olive oil enrichment. Enzyme had an optimal activity at 37°C and pH 7.0, and sodium dodecyl sulfate increased lipase activity. NH ₄NO₃ increased enzyme production, whereas organic nitrogen had no effect. The effect of the type of carbon sources on lipolytic enzyme production was also studied. The best results were obtained with starch and maltose (148,932 and 141,629 U/g, respectively), whereas a rather low enzyme activity was found in cultures grown on glucose and galactose (approx 118,769 and 123,622 U/g, respectively). Enzyme was inhibited with Mn⁺² and Ni⁺² by 68 and 74%, respectively. By contrast, Ca⁺² enhanced enzyme production by 5%.     

A statistical approach using L<Subscript>25</Subscript> orthogonal array method to study fermentative production of clavulanic acid by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> MTCC 1142

Clavulanic acid is a naturally occurring antibiotic produced by Streptomyces clavuligerus. The present work reports on clavulanic acid production by Streptomyces clavuligerus MTCC 1142 using one-factor-at-a-time and L₂₅ orthogonal array. The one-factor-at-a-time method was adopted to investigate the effect of media components (i.e., carbon source, nitrogen source and inoculum concentration) and environmental factors such as pH for clavulanic acid production. Production of clavulanic acid by Streptomyces clavuligerus was investigated using seven different carbon sources (viz. glucose, sucrose, modified starch, rice-bran oil, soybean oil, palm oil, and glycerol) and six different nitrogen sources (viz. peptone, yeast extract, ammonium chloride, ammonium carbonate, sodium nitrate and potassium nitrate). A maximum yield of 140 μg/mL clavulanic acid was obtained in the medium containing soybean oil as a carbon source and yeast extract as nitrogen source. Subsequently, the concentration of soybean flour, soybean oil, dextrin, yeast extract and K₂HPO₄ were optimized using L₂₅ orthogonal array method. The final optimized medium produced 500 μg/mL clavulanic acid at the end of 96 h as compared to 140 μg/mL before optimization. Synthesis of precursor molecules as a metabolic driving force is of considerable importance in antibiotic synthesis. Attempts to increase the clavulanic acid synthesis by manipulating the anaplerotic flux on C₃ and C₅ precursors by supplementing the medium with arginine, ornithine, proline, valine, leucine, isoleucine, pyruvic acid and á-ketoglutarate were successful. Supplementing the optimized medium with 0.1 M arginine and 0.1 M leucine increased the yield of clavulanic acid further to 1100 μg/mL and 1384 μg/mL respectively.     

Biosurfactant production by Rhodococcus erythropolis grown on glycerol as sole carbon source

The production of biosurfactant by Rhodococcus erythropolis during the growth on glycerol was investigated. The process was carried out at 28°C in a 1.5-L bioreactor using glycerol as carbon source. The bioprocess was monitored through measurements of biosurfactant concentration and glycerol consumption. After 51 h of cultivation, 1.7 g/L of biosurfactant, surface, and interfacial tensions values (with n-hexadecane) of 43 and 15 mN/m, respectively, 67% of Emulsifying Index (E ₂₄), and 94% of oil removal were obtained. The use of glycerol rather than what happens with hydrophobic carbon source allowed the release of the biosurfactant, originally associated to the cell wall.     

Utilization of Agricultural Residues of Pineapple Peels and Sugarcane Bagasse as Cost-Saving Raw Materials in Scenedesmus acutus for Lipid Accumulation and Biodiesel Production

The aim of this study is to optimize the lipid accumulation in microalgae by using two agricultural residues of pineapple peels and sugarcane bagasse as low-cost organic carbon sources. Green microalgae Scenedesmus acutus was isolated and selected for cultivation. Effects of three initial sugar concentrations and the stage for adding sugar during cultivation on biomass and lipid production were investigated. The results clearly showed that two-stage cultivation is more suitable than one-stage. The maximum biomass concentration and productivity were obtained at 3.85 g/L and 160.42 mg/L/day when sugarcane bagasse was used. The highest lipid content and lipid yield was reached at 28.05 % and 0.93 g/L when pineapple peels were used, while in the case of sugarcane bagasse, 40.89 % and 1.24 g/L lipid content and yield were obtained. Lipid content was found in normal condition (autotrophic) at 17.71 % which was approximately 2.13-fold lower than when sugarcane bagasse was used (40.89 %). Biodiesel production via in situ transesterification was also investigated; the main fatty acids of palmitic acid and oleic acid were found. This work indicates that using agricultural residues as organic carbon sources could be able to increase lipid content and reduce the cost of biofuel production.     

Cultivation of Chlorella protothecoides with Urban Wastewater in Continuous Photobioreactor: Biomass Productivity and Nutrient Removal

The capability to grow microalgae in nonsterilized wastewater is essential for an application of this technology in an actual industrial process. Batch experiments were carried out with the species in nonsterilized urban wastewater from local treatment plants to measure both the algal growth and the nutrient consumption. Chlorella protothecoides showed a high specific growth rate (about 1 day^?1), and no effects of bacterial contamination were observed. Then, this microalgae was grown in a continuous photobioreactor with CO₂–air aeration in order to verify the feasibility of an integrated process of the removal of nutrient from real wastewaters. Different residence times were tested, and biomass productivity and nutrients removal were measured. A maximum of microalgae productivity was found at around 0.8 day of residence time in agreement with theoretical expectation in the case of light-limited cultures. In addition, N-NH₄ and P-PO₄ removal rates were determined in order to model the kinetic of nutrients uptake. Results from batch and continuous experiments were used to propose an integrated process scheme of wastewater treatment at industrial scale including a section with C. protothecoides.     

Adding Value to the Oil Cake as a Waste from Oil Processing Industry: Production of Lipase and Protease by <Emphasis Type="Italic">Candida utilis</Emphasis> in Solid State Fermentation

Olive oil cake is a by-product from the olive oil processing industry and can be used for the lipase and protease production by Candida utilis in solid state fermentation. Different carbon and nitrogen sources were evaluated, and the results showed that the supplementation of the substrate with maltose and starch as carbon sources and yeast extract as a nitrogen source significantly increased the lipase production. The best results were obtained with maltose, whereas rather low lipase and protease activities were found with glucose and oleic acid. Response surface methodology and a five-level–three-factor central composite rotatable design were used to evaluate the effects of the initial moisture content, inoculum size and fermentation time on both lipase and protease activity levels. A lipase activity value of ≈25 U g^-1 and a protease activity value of 110 U g^-1 were obtained under the optimized fermentation conditions. An alkaline treatment of the substrate appeared to be efficient, leading to increases of 39% and 133% in the lipase and protease production, respectively. The results showed that the olive cake could be a good source for enzyme production by solid state fermentation.     

Oil Production Towards Biofuel from Autotrophic Microalgae Semicontinuous Cultivations Monitorized by Flow Cytometry

Two microalgae species (Scenedesmus obliquus and Neochloris oleoabundans) were cultivated in closed sleeve photobioreactors in order to select the best oil producer for further large-scale open raceway pond cultivations, aiming at biofuel production. Scenedesmus obliquus reached a higher maximum biomass concentration (1.41 g l⁻¹) with a lower lipid content (12.8% w/w), as compared to N. oleoabundans [maximum biomass concentration of 0.92 g l⁻¹ with 16.5% (w/w) lipid content]. Both microalgae showed adequate fatty acid composition and iodine values as substitutes for diesel fuel. Based on these results, N. oleoabundans was selected for further open raceway pond cultivations. Under these conditions, N. oleoabundans reached a maximum biomass concentration of 2.8 g l⁻¹ with 11% (w/w) of lipid content. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional gravimetric lipid analysis was found for both microalgae, making this method a suitable and quick technique for the screening of microalgae strains for lipid production and optimization of biofuel production bioprocesses. Medium growth optimization for enhancement of microalgal oil production is now in progress.