Micro-algae come of age as a platform for recombinant protein production

A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins.     

湖北峡东地区九龙湾剖面震旦系陡山沱组微体化石的新发现

中国南方扬子地台震旦系陡山沱组产出丰富的微体化石,它们主要保存在磷块岩以及燧石结核和条带中。文章详细报道湖北峡东地区九龙湾剖面震旦系陡山沱组微体化石,描述以前未曾在峡东地区碳酸盐相燧石中发现的8属8种微体化石。研究九龙湾剖面陡山沱组大型带刺疑源类、微体多细胞藻类和动物胚胎化石的分布特征,发现Tianzhushania spinosa是最早出现的大型带刺疑源类分子,大冰期后微体生物的辐射是一个阶段性渐进的过程。同时．本项研究进一步证实华南扬子区陡山沱组碳酸盐相燧石和磷块岩地层中保存的微体化石面貌基本一致。     

Micro-algae as a source of protein

About five decades ago, the mass production of certain protein-rich micro-algae was considered as a possibility to close the predicted so called "protein gap". Comprehensive analyses and nutritional studies have demonstrated that these algal proteins are of high quality and comparable to conventional vegetable proteins. However, due to high production costs as well as technical difficulties to incorporate the algal material into palatable food preparations, the propagation of algal protein is still in its infancy. To date, the majority of micro-algal preparations are marketed as health food, as cosmetics or as animal feed.     

Potential of a simple solid-phase extraction method coupled to analytical and bioanalytical methods for an improved determination of microcystins in algal samples

Artemia assays and protein phosphatase assays are commonly used for the screening of microcystins (MCs) in algal samples instead of the standard mouse toxicity assay. However, it has been shown that their results are often biased because of the matrix effects. To eliminate the possible interferences in the algal matrices, a new solid-phase extraction (SPE) method using silica gel as a sorbent was developed and evaluated. Results show that this SPE method could not only reduce the toxicity of the Microcystis samples towards brine shrimp by 50-80% but also eliminate 90-100% of the endogenous phosphatase activity from Spirulina and Chlorella samples, thus improving the determination of microcystins in algal samples using either of the two bioanalytical methods. The application of this SPE method as an off-line cleanup for high-performance liquid chromatography (HPLC) with UV detection is also described in this study. After SPE, the HPLC chromatograms of Microcystis samples have clear baselines that have no interferences with the analyte peaks.     

A review of the harvesting of micro-algae for biofuel production

Many researchers consider efficient harvesting is the major challenge of commercialising micro-algal biofuel. Although micro-algal biomass can be ‘energy rich’, the growth of algae in dilute suspension at around 0.02–0.05 % dry solids poses considerable challenges in achieving a viable energy balance in micro-algal biofuel process operations. Additional challenges of micro-algae harvesting come from the small size of micro-algal cells, the similarity of density of the algal cells to the growth medium, the negative surface charge on the algae and the algal growth rates which require frequent harvesting compared to terrestrial plants. Algae can be harvested by a number of methods; sedimentation, flocculation, flotation, centrifugation and filtration or a combination of any of these. This paper reviews the various methods of harvesting and dewatering micro-algae for the production of biofuel. There appears to be no one method or combination of harvesting methods suited to all micro-algae and harvesting method will have a considerable influence on the design and operation of both upstream and downstream processes in an overall micro-algal biofuel production process.     

Effect of different densities of live and dead Chlorella vulgaris on the population growth of rotifers Brachionus calyciflorus and Brachionus patulus (Rotifera)

In order to maintain rotifer populations during periods of low algal production, it is necessary to offer alternate diets, some of which include forms of preserved algae. The present work is based on the effect of live and dead Chlorella vulgaris on the population growth of Brachionus calyciflorus and Brachionus patulus. The experimental design consisted of three algal levels (0.5 x 10(6), 1.5 x 10(6) and 4.5 x 10(6) cells ml-1) offered in three forms (living, frozen and heat-killed). The maximal population density values for B. calyciflorus ranged from 55 +/- 1 ind. ml-1 (at 0.5 x 10(6) cells ml-1) to 471 +/- 72 ind. ml-1 (at 4.5 x 10(6) cells ml-1) with live Chlorella, but was much lower (6 +/- 1 to 26 +/- 6 ind. ml-1) with frozen or heat-killed alga under comparable food levels. However, the maximum population density of B. patulus under live or or heat-killed Chlorella was similar at comparable algal levels but when offered frozen algae it was four times less. The highest mean peak population density was 1,277 +/- 83 ind. ml-1 under 4.5 x 10(6) cells ml-1. The rate of population increase for B. calyciflorus varied from 0.50 to 0.79 using live Chlorella, but under comparable conditions, this range was lower (0.21 to 0.31) for B. patulus. Results have been discussed in light of possible application for aquaculture.     

Identification of an algal carbon fixation-enhancing factor extracted from Paramecium bursaria

The green ciliate Paramecium bursaria contains several hundred symbiotic Chlorella species. We previously reported that symbiotic algal carbon fixation is enhanced by P. bursaria extracts and that the enhancing factor is a heat-stable, low-molecular-weight, water-soluble compound. To identify the factor, further experiments were carried out. The enhancing activity remained even when organic compounds in the extract were completely combusted at 700 degrees C, suggesting that the factor is an inorganic substance. Measurement of the major cations, K+, Ca2+, and Mg2+, by an electrode and titration of the extract resulted in concentrations of 0.90 mM, 0.55 mM, and 0.21 mM, respectively. To evaluate the effect of these cations, a mixture of the cations at the measured concentrations was prepared, and symbiotic algal carbon fixation was measured in the solution. The results demonstrated that the fixation was enhanced to the same extent as with the P. bursaria extract, and thus this mixture of K+, Ca2+, and Mg2+ was concluded to be the carbon fixation-enhancing factor. There was no effect of the cation mixture on free-living C. vulgaris. Comparison of the cation concentrations of nonsymbiotic and symbiotic Paramecium extracts revealed that the concentrations of K+ and Mg2+ in nonsymbiotic Paramecium extracts were too low to enhance symbiotic algal carbon fixation, suggesting that symbiotic P. bursaria provide suitable cation conditions for photosynthesis to its symbiotic Chlorella.     

Optimization for high-density cultivation of heterotrophic Chlorella based on a hybrid neural network model

AIMS: The purpose of this study was to develop a reliable hybrid neural network (HNN) model for heterotrophic growth of Chlorella, based on which optimization for fed-batch (FB) cultivation of Chlorella may be successfully realized. METHODS AND RESULTS: Deterministic kinetic model was preliminarily developed for the optimization of FB cultivation of Chlorella. The highest biomass concentration and the maximum productivity were obtained as: 104.9 g l(-1) dry cell weight and 0.613 g l(-1) h(-1), respectively. After several cultivations had been performed, an HNN model was developed. The efficiency of biomass production was further increased by the optimization using this model. The highest biomass concentration and the maximum productivity attained was: 116.2 g l(-1) dry cell weight and 1.020 g l(-1) h(-1), respectively. CONCLUSION: The HNN model agreed well with experimental results in different cultivations. Comparison between the HNN model and the deterministic model showed that the former had better generalization ability, which made it a reliable tool in modelling and optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: The high cell density and productivity of biomass obtained in this study is of significance for the commercial cultivation of Chlorella. The simple and efficient optimization strategy proposed in this paper may be employed in heterotrophic mass culture of Chlorella as well as other similar organisms.     

Light-scatter analysis of microalgae. Correlation of scatter patterns from pure and mixed asynchronous cultures

A method is described for the first time for rapid and accurate discrimination among several algal types by their light-scattering properties alone. Using a multiangle light-scattering flow system, we obtained light-scatter patterns for individual cells in asynchronous cultures of Chlorella, Chlamydomonas, and Anacystis. The patterns are consistent and distinct for each species. By these signatures, each algal type can be recognized within mixtures.     

Exclusion chromatography with controlled-pore glass beads to isolate Chlorella chromatin and its applications

A simple and rapid method was developed to isolate chromatin from the unicellular alga, Chlorella, by exclusion chromatography utilizing controlled-pore glass beads. This method takes advantage of the giant size of the chromatin supramolecules and does not require the preliminary isolation of cell nuclei. In order to raise the histone yield, commercially available materials were silanized with dimethyldichlorosilane. The isolated algal chromatin had properties similar to those of other organisms, and the histones contained all five components found in calf thymus. A hierarchy of the higher order structures was also observed in the algal chromatin. This method can be used for the study of chromatin in various cell types, especially in microbial cells, from the viewpoints of not only mere preparation but also cell dynamics and fractionation in relation to the specific components or activities. Some application examples are presented.     

The effect of species diversity on lipid production by micro-algal communities

Current research investigating the importance of diversity for biofuel lipid production remains limited. In contrast, the relationship between diversity and productivity within terrestrial and algal primary producers has been well documented in ecology. Hence, we set out to investigate, experimentally, whether diversity may also affect lipid production in micro-algae. We investigated the growth and lipid production of micro-algae using species from all major algal groups. Algae were grown in a large number of treatments differing in their diversity level. Additionally, we compared the growth and lipid production of laboratory communities to natural lake and pond phytoplankton communities of different diversity. Our results show that lipid production increased with increasing diversity in both natural and laboratory micro-algal communities. The underlying reason for the observed ‘diversity–productivity’ relationship seems to be resource use complementarity. We observed higher lipid production of highly diverse algal communities under the same growth and resource supply conditions compared to monocultures. Hence, the incorporation of the ecological advantages of diversity-related resource-use dynamics into algal biomass production may provide a powerful and cost effective way to improve biofuel production.     

A dye binding method for measurement of total protein in microalgae

Protein is a large component of the standing biomass of algae. The total protein content of algae is difficult to measure because of the problems encountered in extracting all of the protein from the cells. Here we modified an existing protein assay to measure total protein in microalgae cells that involves little or no extraction of protein from the cells. Aliquots of fresh or pretreated cells were spotted onto filter paper strips. After drying, the strips were stained in a 0.1% (w/v) solution of the protein stain Coomassie Brilliant Blue R-250 for 16 to 24 h and then destained. The stained protein spots were cut out from the paper, and dye was eluted in 1% (w/v) sodium dodecyl sulfate (SDS). Absorbance at 600 nm was directly proportional to protein concentration. Cells that were recalcitrant to taking up the dye could be either heated at 80°C for 10 min in 1% SDS or briefly sonicated for 3 min to facilitate penetration of the dye into the cells. Total protein measured in Chlorella vulgaris using this method compared closely with that measured using the total N method. Total protein concentrations were measured successfully in 12 algal species using this dye binding method.     

一株重金属镉耐受小球藻的分离鉴定及其生长条件优化

以龙江河镉污染水体预留样本为实验材料,从中筛选得到一株重金属镉耐受藻株,经形态和分子生物学鉴定为小球藻属,命名为Chlorella sp.LQQ-1。实验从温度、pH、Cd²⁺、有机碳源及有机氮源等方面对该藻株生长条件进行优化,并考察其在最佳生长条件下对水体中Cd²⁺的去除效果。结果表明:该小球藻最适宜生长温度为30℃,最适宜pH为7.0左右。Cd²⁺在低浓度下能促进小球藻的生长,当Cd²⁺浓度超过30μmol·L^-1时,小球藻的生长受到抑制。适宜该小球藻生长的最佳葡萄糖添加浓度为10g·L^-1,最佳尿素添加浓度为1g·L^-1。在最佳生长条件下,该小球藻对水体中Cd²⁺的去除率达89.7%。     

Changes of ribulose 1,5-diphosphate carboxylase level during the processes of degeneration and regeneration of chloroplasts in Chlorella protothecoides

RuDP carboxylase was active mainly in chloroplasts and PEP carboxylaseactive principally outside of chloroplasts in Chlorella protothecoides. During the process of chloroplast degeneration in algal cellsinduced by addition of glucose, the activity of RuDP carboxylasesignificantly decreased, whereas the activities of PEP-carboxylaseand -carboxykinase markedly increased. During the process of chloroplast regeneration in "glucose-bleached"algal cells, which contained no detectable amounts of FractionI protein and showed only traces of RuDP carboxylase activity,a light-dependent development of RuDP carboxylase proceededalmost in parallel with the light-induced formation of chlorophyll.The activities of PEP-carboxylase and -carboxykinase, whichwere negligibly low in glucose-bleached cells, developed independentlyof light. Both chloramphenicol and cycloheximide severely inhibited thedevelopment of RuDP carboxylase activity. A relatively low concentrationof glucose also caused a significant suppression. Under theseconditions, chlorophyll formation was inhibited only slightlyby chloramphenicol and very strongly by cycloheximide and glucose. ¹ Deceased, 11 June, 1972. (Received April 25, 1972; )     

药用植物浸提液抑制蛋白核小球藻生长的化感效应

研究了11种药用植物浸提液对水华藻种蛋白核小球藻的影响,结果显示:黄连、重楼、贯众、防己4种药用植物的浸提液均有抑藻效应。当药用植物浸提液的相对浓度为1g/L处理时,其半抑制效应时间LT50的排序为:防己重楼黄连贯众。进一步研究防己的相对浓度梯度抑藻效应,结果表明:在相对浓度为2g/L时,实验3d后的藻细胞几乎全部死亡;防己的抑藻效应受贮藏时间和贮藏温度的影响不显著。在所研究的药用植物中,防己的抑藻效果最好,在抑藻方面有较大应用前景,其它3种药用植物对轻度藻类爆发的控制也有潜在应用价值。     

不同营养条件下原始小球藻对蒽的富集和降解研究

研究了自养与异养条件下原始小球藻对蒽的降解和富集能力 .结果表明 ,自养条件下 ,浓度为1 0mg·L-1的蒽有 48.18%被降解 ,其中 2 8.81%属于自然光降解 ,仅有 19.37%被原始小球藻降解 .而异养条件下的原始小球藻对浓度为 2 .5mg·L-1的蒽降解率达到 33.5 3%,说明异养原始小球藻不仅能耐受高浓度蒽 ,而且表现出比自养原始小球藻更强的蒽降解能力 .两种条件下 ,80 %以上残留的蒽都被富集到藻细胞中 .虽然自养条件下原始小球藻对蒽的生物富集系数达 90 6 4,远大于异养条件下的生物富集系数(1899) ,但异养条件下藻对蒽的绝对富集量 (2 0 2 .2 9μg)远远高于自养条件下的 6 9.6 87μg .     

Effect of iron on growth and lipid accumulation in Chlorella vulgaris

The economic feasibility of algal mass culture for biodiesel production is enhanced by the increase in biomass productivity and storage lipids. Effect of iron on growth and lipid accumulation in marine microalgae Chlorella vulgaris were investigated. In experiment I, supplementing the growth media with chelated FeCl3 in the late growth phase increased the final cell density but did not induce lipid accumulation in cells. In experiment II, cells in the late-exponential growth phase were collected by centrifugation and re-inoculated into new media supplemented with five levels of Fe3+ concentration. Total lipid content in cultures supplemented with 1.2 x 10(-5) mol L(-1) FeCl3 was up to 56.6% biomass by dry weight and was 3-7-fold that in other media supplemented with lower iron concentration. Moreover, a simple and rapid method determining the lipid accumulation in C. vulgaris with spectrofluorimetry was developed.     

A long-wave absorbing form of chlorophyll a responsible for the “red drop” in fluorescence at 298 °K and the F723 band at 77 °K

When Chlorella cells are ruptured at pH 4.6 by sonication in air, its absorption spectrum can be best explained if one assumes that a long-wave chlorophyll a form (Chl a 693) is preferentially destroyed. Using these preparations, and comparing them with the algal suspension and the sonicates prepared at pH 7.8 under argon, we make the following conclusions: (a) The red drop beginning at about 675–680 nm in the action spectrum^* of fluorescence at 298 °K must be due to the presence of a non-(or weakly) fluorescent form of chlorophyll a. We suggest that this form is Chl a 693. The red drop is absent in the aerobic sonicates. (b) The red drop in fluorescence in whole algal cells is not due to any errors in absorption measurements; this drop is clearly present in the anaerobic sonicates. (c) The emission band at 723 nm, discovered by in whole Chlorella cells at 77 °K, may be due to increased fluorescence efficiency of Chl a 693 at low temperature; the F723 band is absent in aerobic sonicates.     

Effects of SO2 and NO on growth of Chlorella sp. KR-1

Effects of the toxic compounds in flue gas, SOx and NOx, on growth of Chlorella sp. KR-1 have been determined. Although growth of KR-1 was suppressed by the toxic compounds, KR-1 exhibited excellent tolerances to SOx compared to other algal strains. When Chlorella KR-1 was cultured with the model gas containing 60 ppm SO2, the linear growth rate was 1.24 g/l day which is about 25% lower than that of the control culture aerated with the gas mixture containing no toxic compounds, SO2 and NO. KR-1 could grow even with the model gas containing 100 ppm SO2 and the linear growth rate of KR-1 in the culture was 0.78 g/l day. The period for lag phase was increased with increasing of SO2 concentration that also resulted in the decrease of the linear growth rate and the maximum cell concentration. Direct CO2 fixation by Chlorella KR-1 has been successfully done using actual flue gases from a liquified natural gas (LNG)- or diesel-fueled boiler. These results indicated that Chlorella KR-1 may be applied for direct CO2 fixation from actual flue gas.