Integration of algae cultivation as biodiesel production feedstock with municipal wastewater treatment: strains screening and significance evaluation of environmental factors

The objectives of this study are to find the robust strains for the centrate cultivation system and to evaluate the effect of environmental factors including light intensity, light–dark cycle, and exogenous CO₂ concentration on biomass accumulation, wastewater nutrient removal and biodiesel production. The results showed that all 14 algae strains from the genus of Chlorella, Haematococcus, Scenedesmus, Chlamydomonas, and Chloroccum were able to grow on centrate. The highest net biomass accumulation (2.01 g/L) was observed with Chlorella kessleri followed by Chlorella protothecoides (1.31 g/L), and both of them were proved to be capable of mixotrophic growth when cultivated on centrate. Environmental factors had significant effect on algal biomass accumulation, wastewater nutrients removal and biodiesel production. Higher light intensity and exogenous CO₂ concentration with longer lighting period promote biomass accumulation, biodiesel production, as well as the removal of chemical oxygen demand and nitrogen, while, lower exogenous CO₂ concentration promotes phosphorus removal.     

光照强度、温度、pH、盐度对小球藻(Chlorella)光合作用的影响

利用测定净光合放氧速率的方法研究了光照强度、温度、pH、盐度对小球藻(Chlorella sp.XQ-200419)和海洋小球藻(Chlorella marina NJ-016)光合作用的影响。小球藻(Chlorella sp.XQ-200419)的适宜光照强度范围为100～1600μmo·lm-2·s-1,光饱和点在500μmo·lm-2·s-1附近;适宜温度范围为25～42.5℃,最适温度为37.5℃;适宜pH值范围为6.5～9.0,最适pH值为7.0;对盐度的适应范围较广,在0～0.6mol/L范围内,随着盐度的升高,净光合放氧速率有下降趋势。海洋小球藻(Chlorella marina NJ-016)的适宜光照强度范围为400～1600μmol·m-2·s-1,光饱和点在1400μmo·lm-2.s-1附近;适宜温度范围为25～42.5℃,最适温度为37.5℃;适宜pH值范围为5.0～9.0,最适pH值为8.0;对盐度有很好的适应性,在0～0.6mol/L范围内,随着盐度升高,净光合放氧速率明显上升。小球藻和海洋小球藻的净光合放氧速率随光照强度、温度、pH值和盐度变化的规律,表明了两种小球藻的基本生理生态学特性:能适应较强的光照强度、较高的温度、中性偏碱的环境和较高的盐度。研究结果有助于小球藻培养条件的优化。两种小球藻对光照强度、温度、pH值和盐度变化的反应也有所不同:与小球藻(Chlorella sp.XQ-200419)相比,海洋小球藻(Chlorella marina NJ-016)对光照强度有更好的适应性,对pH值变化有更宽的适应范围,适当提高盐度对其光合作用有明显的促进作用。这表明海洋小球藻(Chlorella marina NJ-016)在快速生长繁殖方面具有更大的潜力,这一研究结果为筛选适合于大量培养的优良藻种提供了依据。     

三种不同小球藻去除亚硝态氮和氨氮能力的研究

为了研究小球藻在不同质量浓度的氨氮和亚硝态氮环境下的去除能力与生长效果,从实验室挑选3株不同的小球藻CV315-1(Chlorella sp.)、CV315-2(Chlorella sorokiniana)和CV315-3(Chlorella pyrenoidosa)分别置于不同质量浓度的氨氮和亚硝态氮模拟污水中,在温...     

光照强度、温度、pH、盐度对小球藻（Chlorella）光合作用的影响

利用测定净光合放氧速率的方法研究了光照强度、温度、pH、盐度对小球藻（Chlorella sp.XQ-200419）和海洋小球藻（Chlorella marina NJ-016）光合作用的影响。小球藻（Chlorella sp.XQ-200419）的适宜光照强度范围为100～〉1600μmo·lm-2·s-1,光饱和点在500μmo·lm-2·s-1附近;适宜温度范围为25～42.5℃,最适温度为37.5℃;适宜pH值范围为6.5～9.0,最适pH值为7.0;对盐度的适应范围较广,在0～0.6mol/L范围内,随着盐度的升高,净光合放氧速率有下降趋势。海洋小球藻（Chlorella marina NJ-016）的适宜光照强度范围为400～〉1600μmol·m-2·s-1,光饱和点在1400μmo·lm-2.s-1附近;适宜温度范围为25～42.5℃,最适温度为37.5℃;适宜pH值范围为5.0～9.0,最适pH值为8.0;对盐度有很好的适应性,在0～0.6mol/L范围内,随着盐度升高,净光合放氧速率明显上升。小球藻和海洋小球藻的净光合放氧速率随光照强度、温度、pH值和盐度变化的规律,表明了两种小球藻的基本生理生态学特性：能适应较强的光照强度、较高的温度、中性偏碱的环境和较高的盐度。研究结果有助于小球藻培养条件的优化。两种小球藻对光照强度、温度、pH值和盐度变化的反应也有所不同：与小球藻（Chlorella sp.XQ-200419）相比,海洋小球藻（Chlorella marina NJ-016）对光照强度有更好的适应性,对pH值变化有更宽的适应范围,适当提高盐度对其光合作用有明显的促进作用。这表明海洋小球藻（Chlorella marina NJ-016）在快速生长繁殖方面具有更大的潜力,这一研究结果为筛选适合于大量培养的优良藻种提供了依据。     

环境因子对小球藻(Chlorella sp.XQ-20044)光合作用的影响

小球藻(Chlorella sp.XQ-20044)是一株具有应用潜力的产油微藻,本文利用测定净光合放氧速率的方法研究了光照强度、温度、pH值和盐度对其光合作用的影响。研究结果表明:小球藻适宜的光照强度为500~1200μmol·m-2·s-1,光补偿点约30μmol·m-2·s-1,光饱和点在600μmol·m-2·s-1附近;光合作用适宜的温度范围为30~42.5℃,最适温度为40℃;适宜的pH值范围7.0~10.0,最适pH值为8.0;适宜盐度范围0.1~0.3 mol/L,最适盐度为0.2 mol/L。从光合作用特性来看,小球藻能适应较强的光照强度、较高的温度、偏碱性和较高的盐度环境,其中可耐受较高盐度的特性,有助于预防敌害生物的污染,对于实现规模培养,特别是利用开放系统进行规模培养较为有利。     

利用市政污水培养Chlorella vulgaris生产生物柴油

为了考察利用南昌市政污水规模化培养富油微藻生产生物柴油,同时达到净化污水的目的,取南昌市青山湖污水处理厂未经任何处理的市政污水作为普通小球藻(Chlorella vulgaris)生长的培养液。监测了C.vulgaris在市政污水中连续培养10 d的特定生长率、生物质产量以及与之相关的市政污水中氨氮(NH4+-N)、总磷(TP)、化学需氧量(COD)、总悬浮固体(TSS)和挥发性悬浮固体(VSS)的清除情况。实验表明:营养物质的水平显著地影响了C.vulgaris的生长。C.vulgaris的生长率在培养8 d后达到最大,OD680为2.856,总的生物质产量日均最大积累速率为0.01 g/L,油脂含量为干质量的18%,油脂的平均日产量为0.001 g/L。培养10 d内NH4+-N、TP和COD的去除率分别为50.0%、32.1%和26.0%,TSS和VSS的日平均去除速率分别为0.01 g/L和0.006 1 g/L。     

Culture of microalgae Chlorella minutissima for biodiesel feedstock production

Microalgae are among the most promising of non‐food based biomass fuel feedstock alternatives. Algal biofuels production is challenged by limited oil content, growth rate, and economical cultivation. To develop the optimum cultivation conditions for increasing biofuels feedstock production, the effect of light source, light intensity, photoperiod, and nitrogen starvation on the growth rate, cell density, and lipid content of Chlorella minutissima were studied. The fatty acid content and composition of Chlorella minutissima were also investigated under the above conditions. Fluorescent lights were more effective than red or white light‐emitting diodes for algal growth. Increasing light intensity resulted in more rapid algal growth, while increasing the period of light also significantly increased biomass productivity. Our results showed that the lipid and triacylglycerol content were increased under N starvation conditions. Thus, a two‐phase strategy with an initial nutrient‐sufficient reactor followed by a nutrient deprivation strategy could likely balance the desire for rapid and high biomass generation (124 mg/L) with a high oil content (50%) of Chlorella minutissima to maximize the total amount of oil produced for biodiesel production. Moreover, methyl palmitate (C16:0), methyl oleate (C18:1), methyl linoleate (C18:2), and methyl linolenate (C18:3) are the major components of Chlorella minutissima derived FAME, and choice of light source, intensity, and N starvation impacted the FAME composition of Chlorella minutissima. The optimized cultivation conditions resulted in higher growth rate, cell density, and oil content, making Chlorella minutissima a potentially suitable organism for biodiesel feedstock production. Biotechnol. Bioeng. 2011;108: 2280–2287. © 2011 Wiley Periodicals, Inc.     

小球藻藻菌共生体系在产油方面的特性

【目的】研究人工构建藻菌共生体系在产油方面的特性。【方法】从BG11培养基中分离、筛选出无菌小球藻,通过人工共培养方法构建了藻菌共生体系,探讨了共生体系中小球藻的生长及产油特性。【结果】相比无菌小球藻,藻菌共生体系对于藻的生长、油脂积累以及产生生物柴油的脂肪酸组分方面都有明显的促进作用,其中细菌(Stenotrophomonas maltophilia)和小球藻构建的共生体系效果最好,小球藻生物量提高了9%,油脂含量提高了36.3%,C18-1的含量提高了259.2%。【结论】进一步说明人工共培养方法构建藻菌共生体系能够提高微藻生物柴油的质量,具有很好的利用价值。     

利用啤酒废水小球藻异养培养

摘要：【目的】利用小球藻异养培养技术处理啤酒废水,旨在为啤酒废水资源化利用和降低小球藻生产成本提供一个途径。【方法】在含有10 g/L葡萄糖的基本培养基进行异养小球藻高效藻株的筛选,并用于啤酒废水的资源化处理。【结果】从5株小球藻中得到2株适合高密度异养培养的藻株（Chlorella pyrenoidosa 15-2070 和 Chlorella vulgaris 15-2075）,在啤酒废水的资源化处理过程中这2株小球藻得到非常接近的试验结果。利用由废水配制含10 g/L葡萄糖的基本培养液培养Chlorella pyrenoidosa 15-2070获得了5.3 g/L藻细胞;并且在此过程中,啤酒废水得到有效利用,几种主要污染物最高去除率为：CODcr,92.2 %;BOD5,95.1 %;NO3--N,98.5 %;NH4+-N,92.3 %。【结论】啤酒废水中的重要环境污染物在培养小球藻的过程中可以得到有效地清除,并从中可以获得具有商业价值的小球藻细胞。     

Optimization of CO2 concentration and light intensity for biodiesel production by Chlorella vulgaris FACHB-1072 under nitrogen deficiency with phosphorus luxury uptake

Microalgal biodiesel is an alternative bioenergy for the future. Nitrogen deprivation is usually used to increase lipid content in microalgae, however, it also lowers biomass production, resulting in not much increase of lipid productivity. Our previous study found that phosphorus played an important role in enhancing biodiesel productivity of C. vulgaris FACHB-1072 under nitrogen deficient condition. The aim of this study was to optimize two significant parameters of CO₂ concentration (0.03, 4, 6, 12 %) and light intensity (40, 120, 200 μmol photons m^-2 s^-1) with respect to biodiesel productivity and P uptake rate of C. vulgaris FACHB-1072. It was found that the optimized conditions were 4 % CO₂ concentration and 200 μmol photons m^-2 s^-1 light intensity. The maximum biodiesel productivity was 34.56 mg L^-1 day^-1; 2.7 times higher than the control (nutrient sufficient condition). Phosphorus was accumulated as polyphosphate and its maximum uptake rate was 2.08 mg L^-1 day^-1; twice that of the control. After optimization, the performances under nitrogen deficiency were significantly better compared with those under nitrogen sufficiency, which were rarely reported in literature. Our findings suggest a great potential to combine phosphorus removal from wastewater with biodiesel production via microalgae.     

Metabolic profiling reveals growth related FAME productivity and quality of Chlorella sorokiniana with different inoculum sizes

Inoculum size strongly affects cell growth and lipid accumulation of microalgae, one of the most potential biodiesel feedstock, however, the metabolic mechanism(s) of the lipid biosynthesis upon inoculum size has not been fully explored yet. The effects of inoculum size on cell growth, lipid accumulation, and metabolic changes of a green microalga Chlorella sorokiniana were investigated. In our experimental range of inoculum size, the productivity and the cetane number (CN) of fatty acid methyl esters (FAME) increased with increasing initial cell density, and the inoculum of 1 × 10(7) cells mL(-1) processed much higher productivity (up to 2.02-fold) and CN (up to 1.19-fold) of the FAME than the others. A significant correlation between the metabolic profile and quantity and quality of lipid production was revealed by partial least-squares to latent structures (PLS) analysis, and 15 key metabolites were identified. Most of those metabolites were involved in the photosynthetically fixed carbon metabolism. Furthermore, light intensity as one of the vital limitation factors for the high inoculum size cultivation was evaluated by illumination assay and the results revealed that increasing light intensity could improve the polyunsaturated fatty acids composition and lipid accumulation of C. sorokiniana. The lipid productivity of the culture was improved by 71.21% with the light intensity of 110 μmol m(-2) s(-1), compared to that under the irradiance of 65 μmol m(-2) s(-1).     

Glycerolipid synthesis in Chlorella kessleri 11h. I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C16 and C18 acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [14C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C18-sn-2-C16 (C18/C16) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C18-sn-2-C18 (C18/C18) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C18/C18 species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C18/C18 species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

Characterization of a microalga Chlorella sp. well adapted to highly concentrated municipal wastewater for nutrient removal and biodiesel production

The feasibility of growing Chlorella sp. in the centrate, a highly concentrated municipal wastewater stream generated from activated sludge thickening process, for simultaneous wastewater treatment and energy production was tested. The characteristics of algal growth, biodiesel production, wastewater nutrient removal and the viability of scale-up and the stability of continuous operation were examined. Two culture media, namely autoclaved centrate (AC) and raw centrate (RC) were used for comparison. The results showed that by the end of a 14-day batch culture, algae could remove ammonia, total nitrogen, total phosphorus, and chemical oxygen demand (COD) by 93.9%, 89.1%, 80.9%, and 90.8%, respectively from raw centrate, and the fatty acid methyl ester (FAME) content was 11.04% of dry biomass providing a biodiesel yield of 0.12 g-biodiesel/L-algae culture solution. The system could be successfully scaled up, and continuously operated at 50% daily harvesting rate, providing a net biomass productivity of 0.92 g-algae/(L day).     

Studies on uroporphyrinogen decarboxylase from Chlorella kessleri (Trebouxiophyceae, Chlorophyta)

Uroporphyrinogen decarboxylase (UroD) (EC 4.1.1.37) is an enzyme from the tetrapyrrole biosynthetic pathway, in which chlorophyll is the main final product in algae. This is the first time that a study on UroD activity has been performed in a green alga (Chlorella). We isolated and partially purified the enzyme from a Chlorella kessleri (Trebouxiophyceae, Chlorophyta) strain (Copahue, Neuquén, Argentina), and describe for the first time some of its properties. In C. kessleri, the decarboxylation of uroporphyrinogen III occurs in two stages, via 7 COOH and then 6 and 5 COOH intermediates, with the decarboxylation of the 7 COOH compound being the rate-limiting step for the reaction. Cultures in the exponential growth phase showed the highest specific activity values. The most suitable conditions to measure UroD activity in C. kessleri were as follows: 0.23-0.3 mg protein/mL, approximately 6-8 micromol/L uroporphyrinogen III, and 20 min incubation time. Gel filtration chromatography and Western blot assays indicated that UroD from C. kessleri is a dimer of approximately 90 kDa formed by species of lower molecular mass, which conserves enzymatic activity.     

环境因子对小球藻(Chlorella sp. XQ-20044)光合作用的影响

小球藻（Chlorella sp.XQ-20044）是一株具有应用潜力的产油微藻,本文利用测定净光合放氧速率的方法研究了光照强度、温度、pH值和盐度对其光合作用的影响。研究结果表明：小球藻适宜的光照强度为500~1200 μmol·m^-2·s^-1,光补偿点约30 μmol·m^-2·s^-1,光饱和点在600 μmol·m^-2·s^-1附近;光合作用适宜的温度范围为30~42.5℃,最适温度为40℃;适宜的pH值范围7.0~10.0,最适pH值为8.0;适宜盐度范围0.1~0.3 mol/L,最适盐度为0.2 mol/L。从光合作用特性来看,小球藻能适应较强的光照强度、较高的温度、偏碱性和较高的盐度环境,其中可耐受较高盐度的特性,有助于预防敌害生物的污染,对于实现规模培养,特别是利用开放系统进行规模培养较为有利。     

Influence of red light on the activity of phosphofructokinase in Chlorella kessleri

In crude extracts of the unicellular green alga Chlorella kessleri Fott et Novákóva grown in red light the activity of the glycolytic enzyme phosphofructokinase (PFK, EC 2.7.1.11) is about 40% higher compared to white light conditions giving the same dry matter production. Application of cycloheximide and density labelling with D₂O indicate that this increase depends on the de novo synthesis of the enzyme: Twelve h of illumination at a fluence rate of 7 × 10¹⁸ quanta m⁻² s⁻¹ (11.6 μmol m⁻² s⁻¹) suffice to saturate the effect. In autotrophically grown algae maximal increase in enzyme saturate the effect. In autotrophically grown algae maximal increase in enzyme activity is reached in light of 680 nm, while in 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU)-poisoned, glucose-fed cells, light of wavelengths around 727 nm is most effective. Involvement of a phytochrome-like photoreceptor is discussed.     

利用Chlorella vulgaris C9-JN2010去除蓝藻-猪粪沼液废水中氮磷的研究

利用普通小球藻Chlorella vugaris C9-JN2010处理蓝藻-猪粪沼液废水,以实现废水无害化利用。实验考察了氮磷比和沼液浓度对小球藻生长及处理废水效果的影响,结果表明:在氮磷比(20:1)和沼液浓度(5%)条件下培养小球藻,藻细胞生长和废水处理效果最佳,最高细胞干重及生产强度分别为900.1 mg·L~(-1)和85.1 mg·L~(-1)·d~(-1),废水中总氮、总磷、氨氮的去除率分别为84.6%、95.9%和90.5%,对应去除强度分别为5.43 mg·L~(-1)·d~(-1)、0.30 mg·L~(-1)·d~(-1)和4.75 mg·L~(-1)·d~(-1)。利用小球藻可较彻底的去除蓝藻-猪粪沼液废水中氮、磷等营养,达到污水处理效果。     

Simultaneous nutrient removal and lipid production from pretreated piggery wastewater by Chlorella vulgaris YSW-04

The feasibility of using a microalga Chlorella vulgaris YSW-04 was investigated for removal of nutrients from piggery wastewater effluent. The consequent lipid production by the microalga was also identified and quantitatively determined. The wastewater effluent was diluted to different concentrations ranging from 20 to 80 % of the original using either synthetic media or distilled water. The dilution effect on both lipid production and nutrient removal was evaluated, and growth rate of C. vulgaris was also monitored. Dilution of the wastewater effluent improved microalgal growth, lipid productivity, and nutrient removal. The growth rate of C. vulgaris was increased with decreased concentration of piggery wastewater in the culture media regardless of the diluent type. Lipid production was relatively higher when using synthetic media than using distilled water for dilution of wastewater. The composition of fatty acids accumulated in microalgal biomass was dependent upon both dilution ratio and diluent type. The microalga grown on a 20 % concentration of wastewater effluent diluted with distilled water was more promising for generating high-efficient biodiesel compared to the other culture conditions. The highest removal of inorganic nutrients was also achieved at the same dilution condition. Our results revealed the optimal pretreatment condition for the biodegradation of piggery wastewater with microalgae for subsequent production of high-efficient biodiesel.     

Carbon dioxide biofixation by Chlorella vulgaris at different CO2 concentrations and light intensities

In order to develop an effective CO₂ mitigation process using microalgae for potential industrial application, the growth and physiological activity of Chlorella vulgaris in photobioreactor cultures were studied. C. vulgaris was grown at two CO₂ concentrations (2 and 13% of CO₂ v/v) and at three incident light intensities (50, 120 and 180 μmol m^?2 s^?1) for 9 days. The measured specific growth rate was similar under all conditions tested but an increase in light intensity and CO₂ concentration affected the biomass and cell concentrations. Although carbon limitation was observed at 2% CO₂, similar cellular composition was measured in both conditions. Light limitation induced a net change in the growth behavior of C. vulgaris. Nitrogen limitation seemed to decrease the nitrogen quota of the cells and rise the intracellular carbon:nitrogen ratio. Exopolysaccharide production per cell appeared to be affected by light intensity. In order to avoid underestimation of the CO₂ biofixation rate of the microalgae, exopolysaccharide production was taken into account. The maximum CO₂ removal rate (0.98 g CO₂ L^?1 d^?1) and the highest biomass concentration (4.14 g DW L^?1) were determined at 13% (v/v) CO₂ and 180 μmol m^?2 s^?1. Our results show that C. vulgaris has a real potential for industrial CO₂ remediation.     

Removal and biodegradation of nonylphenol by immobilized Chlorella vulgaris

The removal and biodegradation of nonylphenol (NP) by alginate-immobilized cells of Chlorella vulgaris were compared with their respective free cultures. The effects of four cell densities of 10(4) per algal bead were investigated, as were the four algal bead concentrations, with regard to the removal and biodegradation of NP. Although immobilization significantly decreased the growth rate and NP's biodegradation efficiency of C. vulgaris, NP removal over a short period was enhanced. The NP removal mechanism by immobilized cells was similar to that by free cells, including adsorption onto alginate matrix and algal cells, absorption within cells and cellular biodegradation. The optimal cell density and bead concentration for the removal and biodegradation of NP was 50-100×10(4) cells algal bead(-1) and 2-4 beads ml(-1) of wastewater, respectively. These results demonstrated that immobilized C. vulgaris cells under optimal biomass and photoautotrophic conditions are effective in removing NP from contaminated water.