ALGAL BIOREMEDIATION OF THE BERKELEY PIT LAKE

Ongoing research is unraveling the intricacies of the microbial ecology of the Berkeley Pit Lake System, with ever increasing information becoming available regarding the diversity of Algae, Protistans, Fungi and Bacteria that inhabit this mine waste site. Defining the baseline community structure has been the first step not only toward understanding the interactions of the different groups of organisms, but also toward assessing any improvement in biodiversity within the biotic community. Now that this first step has begun, some of these extremophiles, specifically algae, that have been isolated from the Berkeley Pit Lake System are being used as a potential solution for bioremediation. The specific objectives of this research are fivefold: 1) To evaluate the bioremediative potential of our four most rapidly growing species: (Chromulina freiburgensis Dofl., Chlorella ellipsoidea Gerneck, Chlorella vulgaris Beyerinck and Chlamydomonas acidophilla Negoro) in Berkeley Pit Lake System Water with the additions of NaNO₃ and NaPO₄ by using an experimental matrix. This matrix will be used to estimate the minimum nutrient concentrations that would be necessary to achieve the maximum growth of algae and maximum bioremediation of the Berkeley Pit Lake System. 2) To determine which combination of nutrients will stimulate growth of the best bioremediator of our four isolated species in natural Berkeley Pit Lake System waters. In other words, what nutrient combination will give the best bioremediator a competitive edge over the other species. If time permits, different species may be grown in combination to determine if there are synergistic effects (protocooperation) between/among species. 3) To determine a temperature profile for these four species in order to determine their optimal growth temperature in Berkeley Pit Lake System water. 4) To continue to isolate organisms from the Berkeley Pit Lake System and determine their bioremediative potential. 5) Monitor algal and bacterial counts from a profile of Pit Lake System waters. The results to date will be presented for this conference.     

10 Chlamydomonas acidophila from the berkeley pit lake – a strain adapted to an extreme environment

This research was directed to gather a better understanding about the unicellular alga Chlamydomonas acidophila, one of the 6 algal species found in the Berkeley Pit Lake. Berkeley Pit Lake is a flodded, abandoned pit mine with a pH of 2.7 and high metal concentrations. It has been found that the effective concentrations of metals that limit the growth of C. acidophila by 50% were 9.024 mg/L for Cu2+ and 75.4 mg/L for Zn 2+. We have been able to grow C. acidophila from Berkeley Pit samples at high densities in medium containing 15.36 mg/L Cu2+ and 83.65 mg/L Zn2+. Moreover, this species is able to grow in nutrified Berkeley Pit water, which contains approximately 110 mg/L Cu2+ and 323 mg/L of Zn2+. The hypothesis is that the species found in Berkeley Pit Lake represents a genetic strain adapted to high metal concentrations environments. A comparison between the American Type Culture Collection strain of C. acidophila and the strain collected from Berkeley Pit was made. Growth rate of the two strains in Bold Basil Medium, Modified Acid Medium and Berkeley Pit nutrified water were calculated and compared. Moreover, preliminary investigations of the genome of C.acidophila from the Berkeley Pit Lake were initiated.     

9 Beyond biogeography: large-scale patterns of distribution and abundance of intertidal marine algae along the us west coast

This research was directed to gather a better understanding about the unicellular alga Chlamydomonas acidophila, one of the 6 algal species found in the Berkeley Pit Lake. Berkeley Pit Lake is a flodded, abandoned pit mine with a pH of 2.7 and high metal concentrations. It has been found that the effective concentrations of metals that limit the growth of C. acidophila by 50% were 9.024 mg/L for Cu2+ and 75.4 mg/L for Zn 2+. We have been able to grow C. acidophila from Berkeley Pit samples at high densities in medium containing 15.36 mg/L Cu2+ and 83.65 mg/L Zn2+. Moreover, this species is able to grow in nutrified Berkeley Pit water, which contains approximately 110 mg/L Cu2+ and 323 mg/L of Zn2+. The hypothesis is that the species found in Berkeley Pit Lake represents a genetic strain adapted to high metal concentrations environments. A comparison between the American Type Culture Collection strain of C. acidophila and the strain collected from Berkeley Pit was made. Growth rate of the two strains in Bold Basil Medium, Modified Acid Medium and Berkeley Pit nutrified water were calculated and compared. Moreover, preliminary investigations of the genome of C.acidophila from the Berkeley Pit Lake were initiated.     

食物种类和浓度对壶状臂尾轮虫实验种群动态的影响

使用浓度为0.3mg/mL的椭圆小球藻、尖细栅藻和两者以1:1(湿重比)组成的混合藻在26±1℃下对壶状臂尾轮虫进行单个体培养研究。结果表明,虽然三类食物对轮虫的胚胎发育时间和平均寿命无显著影响,但投喂小球藻时轮虫的生殖前期明显比投喂栅藻或混合藻时短,投喂小球藻时轮虫的生殖期明显比投喂栅藻时长;轮虫的生殖后期历时以栅藻组最长,混合藻组次之,小球藻组最短,三者间具显著差异。轮虫的繁殖率、产卵量和种群内禀增长率均以小球藻组最高,混合藻组次之,栅藻组最低。由此可见,小球藻是该种轮虫培养的最适饵料。以浓度为0.375、0.75、1.5、3.0和5.0×106cells/mL的椭圆小球藻为食物在23±1℃下对壶状臂尾轮虫进行单个体培养研究发现,0.75×106cells/mL是其生存和繁殖的最低浓度阈值;食物浓度对轮虫的胚胎发育时间和生殖后期历时无显著影响;食物浓度为1.5×106cells/mL和3.0×106cells/mL时,轮虫各主要发育阶段的历时和产卵量间也无显著差异;食物浓度高于3.0×106cells/mL或低于1.5×106cells/mL对轮虫的生殖前期无显著影响,但使轮虫的平均寿命和产卵量显著减小;食物浓度高于3.0×106cells/mL时,轮虫的生殖期显著缩短。轮虫种群的繁殖率、净生殖率和内禀增长率皆以3.0×106cells／mL组最高,1.5×106cells／mL组次之,0.75×106 cells／mL和5.0×106cells／mL组较低。因此,轮虫种群增长的适宜食物浓度范围为1.5-3.0×106cells／mL,最适食物浓度是3.0×106cells／mL。       

RETENTION OF DISSOLVED COMPOUNDS BY MEMBRANE FILTERS AS AN ERROR IN THE 14C METHOD OF PRIMARY PRODUCTION MEASUREMENT1

Membrane filters retain ¹⁴C bicarbonate, ¹⁴C glycollate, and other ¹⁴C labeled substances from filtrates of algal cultures and lake water. By refiltering different volumes of the filtrate from algal cultures and from lake water after incubation with ¹⁴C bicarbonate, it was shown that the labeled material retained was not proportional to volume but showed a saturation effect with increasing volume filtered. When the radioactivity retained by a filter is divided by the volume filtered, decreasing values are obtained with increased volume filtered. This radioactivity may represent a significant addition to the radioactivity in particulate material on the filters, resulting in a similar type of curve when different volumes of lake water or cultures are filtered. Values of radioactivity per milliliter were constant using Chlorella in Chu 10 medium. However, the curve could, be obtained by increasing pH and bicarbonate concentrations in the medium and on resuspending the algae in Lake Ontario (winter) filtrate. The values of cpm/ml retained from filtrates were low in Cryptomonas cultures and the curve was not obtained unless population density was reduced, thus increasing the relative contribution to the radioactivity on the filters. The curve was not always obtained in lake water. It was significant in 10 out of 14 experiments in Lake Ontario and in 2 out of 5 experiments in Grenadier Pond. Changes in lake water rather than in experimental techniques were probably responsible. On 2 occasions when values of cpm/ml were constant in Lake Ontario, addition of sodium bicarbonate without a pH change resulted, in a significant curve. Our experiments do not disprove the possibility of cell damage during Millipore filtration, however, it has been shown that ¹⁴C labeled substances retained from solutions can account for the entire range of decreasing values as a function of volume filtered.     

Glutamate dehydrogenases of unicellular green algae: effects of nitrate and ammonium in vivo]

The constitution and control by the inorganic nitrogen source of glutamate dehydrogenases of some unicellular green algae have been studied. The Ankistrodesmus braunii and Scenedesmus obliquus cells contain two different glutamate dehydrogenases, one of which is NADP-specific, the other is active with both NAD and NADP. Their synthesis does not depend on the nitrogen source. The activity of NADP-specific glutamate dehydrogenase increases sharply during nitrogen starvation. In Chlorella pyrenoidosa 82 and Ch. ellipsoidea only one constitutive double specific glutamate dehydrogenase is observed. Its activity does not change depending on the nitrogen nutrition conditions. In the cells of the thermophylic Chlorella strain Chlorella sp. K. ammomium induces a de novo synthesis of NADP-specific glutamate dehydrogenase in addition to the constitutive double specific glutamate dehydrogenase. Thus, the algae tested contain constitutive double specific glutamate dehydrogenase. The NADP-specific enzyme is absent in two Chlorella strains, is constitutive in A. braunii and S. obliquus, and is ammonium-inducible in three thermophylic Chlorella strains.     

Effect of Filtered Cultures of Flagellate Ochromonas sp. on Colony Formation in Microcystis aeruginosa

The possible effect of filtered cultures of flagellate Ochromonas sp. on colony formation in M. aeruginosa was investigated in this paper. The results show that filtered cultures of flagellates fed with M. aeruginosa could induce colony formation in M. aeruginosa. Furthermore, induction strength is clearly dependent on the concentration of flagellates and filtered cultures. However, no colonial M. aeruginosa was found in the treatments of filtered cultures of flagellates fed with Microcystis wesenbergii, filtered cultures of flagellate fed with Chlorella pyrenoidosa, and algae homogenates. This suggests that infochemicals released from flagellates fed with M. aeruginosa may be a trigger for colony formation in M. aeruginosa. The clearance rates of flagellates on algae were markedly decreased when they were cultivated with induced colonial M. aeruginosa. These indicate that colony formation in M. aeruginosa is a predator‐induced defense which could reduce predation risk from flagellates (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

纳米铜粉对浮游植物生长的影响

纳米金属粉末产品在医学、环保和生物工程方面的广泛应用,引发人们对其安全性问题的日益增长的关注,但目前这方面的研究成果十分缺少。实验以斜生栅藻、椭圆小球藻、四列藻为实验对象探讨纳米铜粉和普通铜粉对微藻生长的影响,实验结果表明两种粒径的铜粉对微藻的生长存在不同程度的抑制效应,三种微藻的细胞密度和叶绿素a含量的增加与铜粉的加入量负相关,2.0mg·L^-1纳米铜粉就可以完全抑制微藻生长,椭圆小球藻对纳米铜粉的耐受力相对斜生栅藻、四列藻而言最弱,1.3mg·L^-1纳米铜粉培养条件下的椭圆小球藻即大量死亡。纳米铜粉比相同含量甚至十倍百倍于其含量的普通铜粉对几种微藻的抑制效应要大得多(p<0.05),33.3mg·L^-1和333.3mg·L^-1普通铜粉培养条件下的斜生栅藻和83.3mg·L^-1普通铜粉培养条件下的椭圆小球藻还可以缓慢生长,但3.3mg·L^-1纳米铜粉培养条件下的斜生栅藻和8.3mg·L^-1纳米铜粉或833.3mg·L^-1普通铜粉培养条件下的椭圆小球藻生长几乎停止。     

The effect of light on nitrate and nitrite assimilation by chlorella and ankistrodesmus

Light stimulates the assimilation of nitrate and nitrite by two green algae, Chlorella pyrenoidosa and Ankistrodesmus braunii. Assimilation can be observed when the algae are illuminated in the absence of carbon dioxide under both aerobic and anaerobic conditions. The rates of assimilation by Chlorella do not depend on the presence of carbon dioxide, but Ankistrodesmus assimilates nitrate and nitrite more rapidly when cultures are illuminated in the presence of carbon dioxide than in its absence. The ratios of O(2) : NO(3') and O(2) : NO(2') vary from one experiment to the other and, with the exception of Chlorella cultures reducing nitrite they are higher than the 'expected' values of 2.0 and 1.5 respectively. Oxygen evolution accompanying nitrate and nitrite by algae illuminated in the absence of carbon dioxide is completely inhibited by DCMU at concentrations of 4 × 10(-6) M. However, nitrite assimilation by both Ankistrodesmus and Chlorella and nitrate assimilation by Ankistrodesmus are less sensitive to the inhibitor.     

Studies on the purification of wastewater from the nitrogen fertilizer industry by intensive algal cultures. I. Growth of Chlorella vulgaris in wastes.

The possibility of growth of intensive cultures of Chlorella vulgaris on industrial wastewater from nitrogen fertizer plant containing ammonia, urea and nitrate was investigated. Good growth of algae was obtained when the waste was enriched with phosphorus and inoculum contained a high number of cells. The optimal pH for the culture was 7.0--8.0. The main factor limiting growth of algae on wastes on the concentration of ammonia nitrogen. Chlorella vulgaris grows quite well in wastes containing 600 mg NH4-N/l but is inhibited at concentration about 100 mg NH4-N/l.     

小球藻高密度培养及油脂提取条件的优化

【目的】高密度培养小球藻及优化油脂提取条件。【方法】通过进行单因素实验研究不同培养基组成及环境因子对其细胞生长影响,并采用超声波提取法进行正交实验对藻粉油脂提取条件进行研究。【结果】对椭圆小球藻Y4进行异养培养,最适培养条件为:葡萄糖50 g/L,硝酸钾2 g/L,适宜的培养温度、摇床转速和接种量分别为29°C、180 r/min和20%。在此基础上,进行了1 L发酵罐培养实验,获得了干重18.25 g/L的生物量。通过对油脂提取条件进行优化,Y4的油脂提取率由优化前的25.0%提高到60.2%,提高了35.2%。【结论】优化了小球藻的培养条件及油脂提取条件,促进了小球藻的开发和利用。     

一株太湖水域蓝藻噬藻体的分离与鉴定

研究首次报道在太湖筛选到的一株感染铜绿微囊藻的噬藻体。在太湖蓝藻水华暴发区域采集水样, 经0.22 μm 微孔滤膜过滤、超滤浓缩后感染对数期的不同株微囊藻, 对感染效果明显的进行进一步研究。研究发现M. aernginosa 905 有明显感染, 用CsCl 不连续密度梯度离心的方法对噬藻体进行纯化并研究噬藻体的一步生长曲线。研究发现: 在MOI=10-5 的感染条件下, 该噬藻体感染M. aernginosa 的潜伏期为2h, 裂解期为4—6h, 稳定期为6—12h, 裂解量为4 pfu/cell。透射电镜观察此噬藻体头部为二十面体, 直径约50 nm, 具一很短尾部。此外在不加任何保护剂的情况下, 此噬藻体在-20℃和-80℃下保存感染力丧失, 但在4℃条件下保存, 其感染活性可维持50d 以上。研究为探讨用噬藻体控制蓝藻水华提供了重要基础       

BIOCHEMICAL TAXONOMY AND MOLECULAR PHYLOGENY OF THE GENUS CHLORELLA SENSU LATO (CHLOROPHYTA)

A multimethod approach was used to characterize unicellular green algae that were traditionally assigned to the genus Chlorella Beijerinck and to resolve their phylogenetic relationships within the Chlorophyta. Biochemical, physiological, and ultrastructural characters, together with molecular data such as DNA base composition and DNA hybridization values, were compared with a molecular phylogeny based on complete 18S rRNA sequences. Our results show that Chlorella taxa are dispersed over two classes of chlorophytes, the Trebouxiophyceae and the Chlorophyceae. We propose that only four species should be kept in the genus Chlorella (Chlorophyta, Trebouxiophyceae): C. vulgaris Beijerinck, C. lobophora Andreyeva, C. sorokiniana Shih. et Krauss, and C. kessleri Fott et Nováková. Common characteristics of these taxa are glucosamine as a dominant cell wall component and the presence of a double thylakoid bisecting the pyrenoid matrix. Norspermine, norspermidine, and secondary carotenoids are never produced. Other "Chlorella" species belong to different taxa within the Trebouxiophyceae ( "C." protothecoides = Auxenochlorella protothecoides [Krüger] Kalina et Punčochářová, "C." ellipsoidea, "C." mirabilis, "C." saccharophila, and "C." luteoviridis ) and Chlorophyceae ( "C." zofingiensis and "C." homosphaera = Mychonastes homosphaera Kalina et Punčochářová). The latter taxa can easily be recognized by the production of secondary carotenoids under nitrogen-deficient conditions.     

人工滩涂湖泊滴水湖浮游藻类群落特征

于2009年3月—2010年2月逐月对中国最大人工滩涂湖泊——滴水湖的浮游藻类群落特征进行研究。利用CANOCO4.5软件对浮游藻类数据和环境因子数据进行典范对应分析(CCA),以揭示浮游藻类对生态环境的响应。结果表明:全年共检测到浮游藻类186种及变种,隶属于8门92属,其中绿藻门种类最多,其次是蓝藻门和硅藻门;主要优势种为小席藻、微小平裂藻、银灰平裂藻、不定微囊藻、弱细颤藻、啮蚀隐藻、四尾栅藻、扁圆卵形藻;藻类的年均丰度和年均生物量分别为(4552.29±4591.33)×104cells·L-1和8.15±6.63mg·L-1,藻类现存量各月份间差异极显著(P<0.01),但站点间差异不显著;多样性分析显示,藻类物种多样性较好;典范对应分析表明,水温、总氮等是影响滴水湖藻类群落结构的主要因素。     

Critical problems with extraction of ATP for bioluminescence assay of plankton biomass

Recovery of ATP by boiling tris extraction was 90–95 percent greater in 1 liter grab samples than in concentrated net samples. ATP losses were attributed to insulating effects promoted by accumulation of detritus on filters. A series of extractions over a concentration range of whole or size-segregated plankters and cultured algae was made to determine volume of water to be filtered for optimum extraction efficiency. Accuracy of ATP assays was optimized by: (i) using large diameter (i.e. 47 mm) acetate filters; (2) limiting sample volume filtered to 50 ml when particulate organic carbon (POC) exceeded 0.4 mg l^–1; and (3) performing extractions in boiling tris maintained initially on a laboratory hot plate at 400°C as opposed to hot water bath at 100°C.Additional problems were encountered in using published cellular carbon: ATP ratios for conversion of ATP data to biomass as carbon. Ratios of POC: ATP in cultures of sheathed blue-green algae reached 550 : i, while non-sheathed forms yielded ratios near values previously reported for plankton communities. Difficulties in applying a uniform conversion factor may be expected in plankton communities containing significant volumes of sheathed blue-greens.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase gene expression and diversity of Lake Erie planktonic microorganisms.

Carbon dioxide fixation is carried out primarily through the Calvin-Benson-Bassham reductive pentose phosphate cycle, in which ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme. The primary structure of the large subunit of form I RubisCO is well conserved; however, four distinct types, A, B, C, and D, may be distinguished, with types A and B and types C and D more closely related to one another. To better understand the environmental regulation of RubisCO in Lake Erie phytoplanktonic microorganisms, we have isolated total RNA and DNA from four Lake Erie sampling sites. Probes prepared from RubisCO large-subunit genes (rbcL) of the freshwater cyanobacterium Synechococcus sp. strain PCC6301 (representative of type IB) and the diatom Cylindrotheca sp. strain N1 (representative of type ID) were hybridized to the isolated RNA and DNA. To quantitate rbcL gene expression for each sample, the amount of gene expression per gene dose (i.e., the amount of mRNA divided by the amount of target DNA) was determined. With a limited number of sampling sites, it appeared that type ID (diatom) rbcL gene expression per gene dose decreased as the sampling sites shifted toward open water. By contrast, a similar trend was not observed for cyanobacterial (type IB) rbcL gene expression per gene dose. Complementary DNA specific for rbcL was synthesized from Lake Erie RNA samples and used as a template for PCR amplification of portions of various rbcL genes. Thus far, a total of 21 clones of rbcL genes derived from mRNA have been obtained and completely sequenced from the Ballast Island site. For surface water samples, deduced amino acid sequences of five of six clones appeared to be representative of green algae. In contrast, six of nine sequenced rbcL clones from 10-m-deep samples were of chromophytic and rhodophytic lineages. At 5 m deep, the active CO2-fixing planktonic organisms represented a diverse group, including organisms related to Chlorella ellipsoidea, Cylindrotheca sp. strain N1, and Olisthodiscus luteus. Although many more samplings at diverse sites must be accomplished, the discovery of distinctly different sequences of rbcL mRNA at different water depths suggests that there is a stratification of active CO2-fixing organisms in western Lake Erie.     

Variant forms of a group I intron in nuclear small-subunit rRNA genes of the marine red alga Porphyra spiralis var. amplifolia

A group IC1 intron occurs in nuclear small-subunit (18S) ribosomal RNA (SSU rRNA) genes of the marine red alga Porphyra spiralis var. amplifolia. This intron occurs at the same position as the self- splicing group IC1 introns in nuclear SSU rDNAs of the fungus Pneumocystis carinii and in the green alga Chlorella ellipsoidea and shares sequence identity with the Pneumocystis carinii intron in domains L1, P1, P2, and L2, outside the conserved core. Three size variants, differing in amount of sequence in L1, exist and are differentially distributed in geographically distinct populations. Preliminary data suggest that the largest variant can self-splice in vitro. Short open reading frames are present but do not correspond to known genes. Repeated nucleotide motifs, reminiscent of duplicated target sites of transposons or Alu elements, are associated with the intron and with one of the variant forms of L1. Insertions are present in nuclear SSU rDNAs of several other Porphyra species and of the red alga Bangia atropurpurea; insertionless rDNA variants also occur in several Porphyra species. Our observations are most readily explained by intron mobility, although it remains unclear how transfer could have been mediated between genomes of organisms as ecologically diverse as marine red algae, freshwater green algae, and a mammalian-pathogenic fungus.      

以异养细胞作为种子的椭圆小球藻产油脂光自养培养优化

异养细胞种子/光自养培养方法是一种可异养培养的能源微藻培养的有效方法,但已有文献尚未从工艺优化角度考察其发展潜力。为了获得较高细胞密度的用于光自养培养的种子和提高光自养培养的细胞密度与油脂产率,对异养细胞种子/光自养培养的培养基和培养条件进行了优化。结果表明,采用优化后的培养基,椭圆小球藻在摇瓶中异养培养的最高藻细胞密度可达11.04 g/L,比在初始培养基条件下提高了28.0%,在5 L发酵罐中异养培养的藻细胞密度达到73.89 g/L;在2 L柱式光生物反应器中光自养培养的藻细胞密度、油脂含量和油脂产率分别达1.62 g/L、36.34%和6.1 mg/(L·h),油脂成分主要为含C16-C18碳链的脂肪酸,是制备生物柴油的理想原料。经过优化,异养细胞种子/光自养培养这一方法能够显著地提高椭圆小球藻产油脂的能力,这进一步表明异养细胞种子/光自养培养方法有望成为可异养的能源微藻的高效培养方式。     

SALINITY AND NUTRIENT LIMITATIONS ON GROWTH OF BENTHIC ALGAE FROM TWO ALKALINE SALT LAKES OF THE WESTERN GREAT BASIN (USA)1

Enrichment cultures of littoral benthic algae from Mono Lake, California, and Abert Lake, Oregon, were grown under conditions of varied salinity and nutrient content. Field-collected inocula were composed mainly of diatoms and filamentous blue-green and green algae. The yield of long-term cultures (30 days) showed tolerance over a broad salinity range (50–150 g·L^?1) for Mono Lake-derived algae. Algae from Abert Lake had a lower range of tolerance (25–100 g·L^?1) Organic content and pigment concentrations of algae from both lakes were also reduced above the tolerated salinity level. Within the range of salinity tolerance for Mono Lake algae, initial growth rates and organic content were reduced by increased salinity. The effects of macro- and micronutrient enrichment on algal growth in Mono Lake water were also tested. Only nitrogen enrichment (either as ammonium or nitrate) stimulated algal growth. Although the benthic algae cultured here had wide optima for salinity tolerance, the rates of growth and storage were limited by increased salinity within the optimum range. Although the lakes compared had similar species composition, the range and limits of tolerance of the algae were related to salinity of the lake of origin.