“Rational” Management of Dichlorophenols Biodegradation by the Microalga Scenedesmus obliquus

The microalga Scenedesmus obliquus exhibited the ability to biodegrade dichlorophenols (dcps) under specific autotrophic and mixotrophic conditions. According to their biodegradability, the dichlorophenols used can be separated into three distinct groups. Group I (2,4-dcp and 2,6 dcp – no meta-substitution) consisted of quite easily degraded dichlorophenols, since both chloride substituents are in less energetically demanding positions. Group II (2,3-dcp, 2,5-dcp and 3,4-dcp – one meta-chloride) was less susceptible to biodegradation, since one of the two substituents, the meta one, required higher energy for C-Cl-bond cleavage. Group III (3,5-dcp – two meta-chlorides) could not be biodegraded, since both chlorides possessed the most energy demanding positions. In general, when the dcp-toxicity exceeded a certain threshold, the microalga increased the energy offered for biodegradation and decreased the energy invested for biomass production. As a result, the biodegradation per cell volume of group II (higher toxicity) was higher, than group I (lower toxicity) and the biodegradation of dichlorophenols (higher toxicity) was higher than the corresponding monochlorophenols (lower toxicity). The participation of the photosynthetic apparatus and the respiratory mechanism of microalga to biodegrade the group I and the group II, highlighted different bioenergetic strategies for optimal management of the balance between dcp-toxicity, dcp-biodegradability and culture growth. Additionally, we took into consideration the possibility that the intermediates of each dcp-biodegradation pathway could influence differently the whole biodegradation procedures. For this reason, we tested all possible combinations of phenolic intermediates to check cometabolic interactions. The present contribution bring out the possibility of microalgae to operate as “smart” bioenergetic “machines”, that have the ability to continuously “calculate” the energy reserves and “use” the most energetically advantageous dcp-biodegradation strategy. We tried to manipulate the above fact, changing the energy reserves and as a result the chosen strategy, in order to take advantage of their abilities in detoxifying the environment.     

Harvesting of Scenedesmus obliquus in wastewaters: Auto- or bioflocculation?

Autoflocculation and bioflocculation are considered to be the most promising means for the economical harvesting of microalgae. We have therefore studied these phenomena with cultures of Scenedesmus obliquus produced during biological tertiary wastewater treatment. The quantity of extracellular polymers produced during ageing of the cultures proved insufficient to initiate bio-flocculation while the concentration of Ca(2+) and PO(4) (3-) of the treated effluent were too low to induce autoflocculation. It has been shown, however, that the algae sediment more readily upon ageing, possibly as a result of increased ceil density. The use of density gradients made with Percoll (a colloidal solution of silica particles) allowed measurement of the true cell density and showed that this increases when cultures enter the declining growth phase. The quality of the biomass thus harvested is, however, considerably impaired, protein content decreasing from 62.7% (dry wt) during the exponential growth phase (day 5) to 14% at the end of cultures (day 21).     

Unterschiedliche Wirkung von Captan auf das Wachstum einiger Stämme von Chlorella und Scenedesmus

Zusammenfassung Das Fungicid Captan erwies sich bei 37 Stämmen von Chlorella als ein wirksamer Wachstumshemmstoff. Nur 3 Chlorella-Stämme besitzen eine gewisse Captanresistenz. Das Merkmal Captanresistenz scheint in der Gattung Chlorella nicht artspezifisch verteilt zu sein.Bei Scenedesmus acutus f. alternans Hortob. und Scenedesmus armatus (Chod.) Smith wird Captan noch in einer Dosis von 50 mg/l vertragen, ohne daß Anzeichen einer Hemmwirkung zu erkennen sind.

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Differential action of captan on the growth of some strains of Chlorella and Scenedesmus

Summary The fungicide captan was found to inhibit strongly the photoautotrophic growth of 37 Chlorella strains. Only 3 strains are fairly resistant to captan. In the genus Chlorella resistance to captan does not seem to be species specific.Two strains of Scenedesmus (Scenedesmus acutus f. alternans Hortob. and Scenedesmus armatus (Chod.) Smith) tolerate captan up to 50 mg/l without being inhibited at all.

     

S-Adenosyl-l-methionine–cycloartenol methyltransferase activity in cell-free systems from Trebouxia sp. and Scenedesmus obliquus

1. Homogenates prepared from Trebouxia sp. 213/3 and Scenedesmus obliquus exhibited S-adenosyl-l-methionine-cycloartenol methyltransferase activity. 2. The products of the reaction, with cycloartenol as the substrate, were 24-methylenecycloartanol and cyclolaudenol. 3. Optimal enzyme activity was found in homogenates prepared at pH7.6 and the transmethylase was distributed between the supernatant and microsomal fractions of the Trebouxia homogenate. 4. The relevance of these results is discussed in relation to C(28) and C(29) sterol production in the algae.     

盐度对一株淡水栅藻Scenedesmus sp.生长及生化组成的影响

通过管式光生物反应器培养,分析比较不同盐度(0-40‰)对一株淡水栅藻(Scenedesmus sp.)生长和生化特性影响,评价海水培养该藻株的产业化潜力,探讨其高盐适应机制,为进一步海水驯化研究提供理论参考。采用显微观察、索氏提取法、凯氏定氮法、苯酚硫酸法、气相色谱法及高效液相色谱法分别测定微藻生长及蛋白质、油脂、多糖含量以及色素、脂肪酸组成情况。结果表明,高盐条件下藻细胞明显增大,并出现自然沉降现象,可实现低成本采收。随盐度增加,栅藻生长逐步被抑制,40‰盐度条件下生长完全停止,但在3‰盐度下仍良好生长,培养末期生物量可达2.84 g/L。30‰盐度组蛋白质含量相对淡水组提高95.40%、产率与淡水组相近,加之高盐培养可较大幅度降低蛋白质的生产成本,因此,采用海水培养栅藻来开发蛋白资源具有一定的潜力。研究还表明,30‰盐度下藻细胞内β-胡萝卜素、虾青素、可溶性多糖等成分大量积累,可能是其适应高盐胁迫的重要生理基础。     

组合处理对新型斜生栅藻(Scenedesmus obliqast)积累虾青素的研究

[目的]研究20μmol/L褪黑素和0.2 mg/L水杨酸(SA)组合3.0 mg/L茉莉酸甲酯等组合处理诱导新型斜生栅藻协同促进积累虾青素的效应。[方法]在优化和改良的BG11培养基中培养,营养细胞培养阶段用一系列平行的40.0W红光灯管和1.2 m长28.0 W蓝光灯管照射藻液(总光照强度为252μmol·m~(-2)s~(-1)),连续培养至对数生长期,每天震荡摇动培养物2次,防止黏壁。对数生长期1 d后,通过1.2 m长蓝光灯管+高光强+氮饥饿+51.8 g/L葡萄糖和20.0 g/L木糖,进行生物量发酵和诱导部分虾青素,继续培养3 d(方案1),然后在广泛诱导阶段,分步提高光照强度高约555μmol·m~(-2)s~(-1)后,培养3 d(方案2),继续光照,再通过2μmol/L褪黑素组合3.0 mg/L茉莉酸甲酯加0.2 mg/L水杨酸诱导获得虾青素,继续培养5 d(方案3)。[结果]3个处理方案均能轻微促进斜生栅藻细胞密度的增加,最大生物量达到0.91×10~6个/mL强度。方案3处理组藻细胞内SA含量适度提升为1.0 mmol/L,藻体内虾青素含量为9.6%,显著高于对照组(P 0.05)。运用荧光定量PCR分析表明:经过方案3处理后,斜生栅藻新藻种(Scenedesmus obliqast)体内ipi、psy、crtR-b基因的表达量分别为对照的15.60、16.20、19.68倍,均有显著差异(P 0.05)。尤其是lyc基因的表达量为对照的31.20倍,达到差异极显著(P 0.01)。从方案1到方案3,bkt、zds和pds基因的表达量逐渐增加,但不明显(P 0.05)。[结论]方案3的组合处理能使藻细胞内积累适度提升的水杨酸信号分子,适度提升的胞内SA含量就显著促进了虾青素的增长,而且组合处理协同调控7种关键酶基因表达。因此,2μmol/L褪黑素组合0.2 mg/L水杨酸加3.0 mg/L茉莉酸甲酯等诱导是非常有效的。     

Accumulation,degradation and biological effects of lindane on Scenedesmus obliquus (Turp.) Kütz

A study was made of accumulation, degradation and biological effects of lindane (r-BHC) on Scenedesmus obliquus (Turp.) Kütz. The work was conducted in a sewage reservoir in Han-Ku, Tianjin. Experimental results show that at 0.1–10 mgl⁻¹ concentration of lindane, the growth, chlorophyll and carotene content in S. obliquus do not differ much from the control. At concentrations of 50–100 mgl⁻¹, however, growth is inhibited, and chlorophyll and carotene content is low. From the beginning of the experiment until the fifth day, growth rate and pigment content increase constantly. On the seventh day, they start to decrease. Spectrographic analysis shows that pigment content decreases, but pigment structure is not changed even at high concentration of lindane. At 1–10 mgl⁻¹ of lindane, amino acids remain unchanged, with the exception of aspartine, arginine, methionine and phenylalanine. Scenedesmus obliquus (Turp.) Kütz possesses a certain accumulating capacity for r-BHC, which is higher at 1 mgl⁻¹ than at 10 mgl⁻¹. Accumulation is enhanced with time and is better under turbulent conditions. Gas chromatograms show that after treatment for 24 h, there are two more peaks at retention time 1′32″ and 1′53″, respectively, suggesting that a certain degree of lindane degradation occurs. The nature of these degradation products awaits further study. As a conclusion S. obliquus (Turp.) Kütz holds promise in the treatment of lindane-containing wastewaters in the range of 1–10 mgl⁻¹.     

两种新型农药对斜生栅藻(Scenedesmus obliquus Ktz.)的毒性研究

LY-04和WSQIF是由华中师范大学农药所合成的两种新型有机磷农药,LY-04属于α-氧代膦酸衍生物,WSQIF属于芳氧基乙酰氧基烃基膦酸脂。选用斜生栅藻(ScenedesmusobliquusKütz.)作为实验生物,通过显微计数测定藻细胞的密度以计算LY-04和WSQIF对斜生栅藻24h、48h、72h、96h的半效应抑制浓度(EC50),研究其对斜生栅藻的毒性效应。结果表明:以丙酮为溶剂时,每隔24h取样,测得LY-04对斜生栅藻的48h、72h、96h的EC50值分别为2942.5mg/L、239.7mg/L和65.0mg/L,而WSQIF的24h、48h、72h、96h的EC50值分别为15.9mg/L、53.0mg/L、98.0mg/L和28.8mg/L。LY-04和WSQIF分别作为杀虫剂和除草剂而研制,经活体研究表明,LY-04具有较强的杀虫活性,WSQIF则具有很强的除草活性,所得EC50值基本符合设计结果。根据毒性分级标准,LY-04和WSQIF的EC50值远大于3mg/L,其对非靶生物中的水生藻类都属于低毒,可推广应用。     

光照强度对于斜生栅藻Scenedesmus obliquus （Turp．）Kuetz．生长的影响

浮游藻类的生长发育,和其他绿色植物一样,与光有密切的关系,因为光直接影响到它的光合作用的进行。在适度的光强度下,光的强度和植物的生长率是成正比的。运方面曾有过不少的工作。但是,在各样光强度的条件下,浮游藻类的数量,     

Oxygen evolution and phosphorylation in Scenedesmus as influenced by the inhibitor-β complex from potato and by phloridzin

Summary Scenedesmus cells were tried as a photosynthesizing test system for the inhibitor- complex from potato (var. Majestic). The main effect on oxygen evolution is an inhibition of the second gush during the induction period. The formation of bound phosphate is increased by the inhibitor when the cell suspensions are shaken in the light, whereas no significant trend is observed in similar experiments in the dark. — Phloridzin inhibits the same part of oxygen evolution as inhibitor- from potato when cells pretreated in light are the test object. Concentrations of 1 mM increase the formation of bound phosphate in light, but at 2 mM there is a decrease in this action. Phloridzin and inhibitor- from potato have different R_f values. — The biochemical effects studied precede possible effects on cell division. — The results are discussed in relation to the localization of the site(s) of action.     

Charakterisierung einer Synchronkultur von Scenedesmus obliquus,ihrer potentiellen Photosyntheseleistung und des Photosynthese-Quotienten während des Entwicklungscyclus

Summary Using synchronous cultures, the change in the potential photosynthetic capacity and the behavior of the photosynthetic quotient were investigated during the life cycle of Scenedesmus obliquus, strain D 3. Scenedesmus obliquus was synchronized under a light-dark regime of 14:10 hours. The quality of synchrony was demonstrated by complete synchronization, homogeneity, exponential growth, shortest possible life cycle and non-susceptibility of the life cycle to the synchronizing procedure. Furthermore, the synchronous culture was characterized by determination of cell number, dry weight, packed cell volume, chlorophylls a and b and the carotenoids during the life cycle.The potential photosynthetic capacity (as O₂ evolution) was measured manometrically and polarographically in white light. This capacity increased from the beginning of the light period until the 8th hour and then declined until the 16th hour, that is until just before release of daughter cell. The percentage difference between the maximum and the minimum of the photosynthetic capacity was the same in the light saturating and light limiting region of photosynthesis. — The photosynthetic quotient was measured by means of Warburg's indirect method. It proved to be constant throughout the life cycle under light saturating and light limiting conditions. The ratio of O₂ evolved to CO₂ consumed was just below unity.The results were compared with those of other authors and their significance was discussed. It was concluded that the change in the photosynthetic capacity is inherent in the normal life cycle of green algae and is not a result of the synchronizing light-dark regime.

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Erster Teil einer Habilitationsschrift der Naturwissenschaftlichen Fakultät der Philipps-Universität Marburg. Auszüge dieser Arbeit wurden auf dem International Congress of Photosynthesis Research in Freudenstadt im Juni 1968 vorgetragen.     

两种新型农药对斜生栅藻（Scenedesmus obliquus Kuetz．）的毒性研究

LY-04和WSQIF是由华中师范大学农药所合成的两种新型有机磷农药,LY—04属于α-氧代膦酸衍生物,WSQIF属于芳氧基乙酰氧基烃基膦酸脂。选用斜生栅藻(Scenedesmus obliquus Kuetz．)作为实验生物,通过显微计数测定藻细胞的密度以计算LY—04和WSQIF对斜生栅藻24h、48h、72h、96h的半效应抑制浓度(EC50),研究其对斜生栅藻的毒性效应。结果表明：以丙酮为溶剂时,每隔24h取样,测得LY—04对斜生栅藻的48h、72h、96h的ECs。值分别为2942．5mg／L、239．7mg／L和65．0mg／L,而WSQIF的24h、48h、72h、96h的EC5。值分别为15．9mg／L、53．0mg／L、98．0mg／L和28．8mg／L。LY—04和WSQIF分别作为杀虫剂和除草剂而研制,经活体研究表明,LY—04具有较强的杀虫活性,WSQIF则具有很强的除草活性,所得EC50。值基本符合设计结果。根据毒性分级标准,LY—04和WSQIF的EC50。值远大于3mg／L,其对非靶生物中的水生藻类都属于低毒,可推广应用。     

Biomass productivity of Scenedesmus dimorphus (Chlorophyceae) was improved by using an open pond–photobioreactor hybrid system

The industrialization of microalgae-based biofuel production has been hampered by low biomass productivity of conventional open ponds. In this research, a hybrid cultivation system that combined an open pond and photobioreactor (PBR), with broth circulating between both, was introduced. The hybrid system was tested under indoor and outdoor conditions using the oleaginous microalgal species Scenedesmus dimorphus. When the PBR(s) in the hybrid system reinforced the light supply to the carbon-replete open pond the biomass reached 1.34 g l^–1, 116% higher than in the non-hybrid system. Subsequent studies showed that higher circulation speed and low volume ratio of PBR vs. open pond would further improve the hybrid effects. When applied outdoors at pilot scale, the biomass productivity of the hybrid system increased 46.3–74.3% compared with the open pond and in September was 12.5% higher than that of PBRs. These results indicate that hybrid cultivation might be a cost-effective way to improve the light usage efficiency of current open pond systems.     

Quantenausbeute und unterschiedliches Verhalten der beiden Photosysteme des Photosyntheseapparates während des Entwicklungsablaufes von Scenedesmus obliquus in Synchronkulturen

Summary Cultures of Scenedesmus obliquus strain D3 synchronized in a light-dark regime of 14:10 hours were used for studies of the photosynthetic activity during the life cycle of the organism.The quantum yield measured throughout the life cycle follows closely the photosynthetic capacity, with a maximum at the 8th and a minimum at the 16th hour in cells whose life cycle was initiated with the beginning of the light period.The amount of photosystem II activity (measured as p-benzoquinone Hill-reaction) and the action spectra demonstrate the same decrease from the 8th to the 16th hour as quantum yield does. However, the reaction of photosystem I seems to be the same throughout the life cycle, when measured as photoreduction or as a part of the light induced absorption change at 520 nm.With support from the observation that the Emerson-enhancement effect is highest at the 8th and lowest at the 16th hour of the life cycle, it was concluded that the two photosystems work with highest activity and in pace at the 8th hour. At the 16th hour the activity of photosystem I remains constant. Since cyclic photophosphorylation is higher at the 16th than at the 8th hour, it is suggested that the capacity of photosystem I, which is freed from photosystem II by a decreased electron flow, is used for additional cyclic photophosphorylation.

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Zweiter, durch einige Versuche ergänzter, Teil einer Habilitationsschrift der Naturwissenschaftlichen Fakultät der Philipps-Universität Marburg. Auszüge dieser Arbeit wurden auf dem International Congress of Photosynthesis Research in Freudenstadt im Juni 1968 vorgetragen (Senger und Bishop, 1969a).     

两种新型农药对斜生栅藻(Scenedesmus obliquus Kütz.)的毒性研究

LY-04和WSQIF是由华中师范大学农药所合成的两种新型有机磷农药,LY-04属于α-氧代膦酸衍生物,WSQIF属于芳氧基乙酰氧基烃基膦酸脂.选用斜生栅藻(Scenedesmus obliquus Kütz.)作为实验生物,通过显微计数测定藻细胞的密度以计算LY-04和WSQIF对斜生栅藻24 h、48 h、72 h、96 h的半效应抑制浓度(EC50),研究其对斜生栅藻的毒性效应.结果表明以丙酮为溶剂时,每隔24 h取样,测得 LY-04对斜生栅藻的48 h、72 h、96 h的EC50值分别为2 942.5 mg/L、239.7 mg/L和65.0 mg/L,而WSQIF的24 h、48 h、72 h、96 h的EC50值分别为15.9 mg/L、53.0 mg/L、98.0 mg/L和28.8 mg/L.LY-04和WSQIF分别作为杀虫剂和除草剂而研制,经活体研究表明,LY-04具有较强的杀虫活性,WSQIF则具有很强的除草活性,所得EC50值基本符合设计结果.根据毒性分级标准,LY-04和WSQIF的EC50值远大于3 mg/L,其对非靶生物中的水生藻类都属于低毒,可推广应用.     

栅藻Scenedesmus sp.蛋白质提取方法优化及氨氮与硝态氮处理下蛋白质组表达差异

旨在寻找一种快速高效提取栅藻Scenedesmus sp.蛋白质的方法并研究氨氮对该藻的毒害机制。使用液氮研磨和超声细胞破碎进行细胞破碎,丙酮沉淀和TCA-丙酮沉淀法进行蛋白纯化,SDS-PAGE进行蛋白样品丰度检验,2-DE电泳技术对差异表达蛋白进行分析。结果显示,对于栅藻Scenedesmus sp.,液氮研磨破碎细胞效果明显优于超声细胞破碎法,前者细胞破碎完全,内容物充分外泄。TCA-丙酮沉淀纯化得到的蛋白样品不仅浓度高,而且丰度和纯度均显著优于丙酮沉淀法。氨氮毒害作用下栅藻Scenedesmus sp.蛋白差异表达分析结果显示85个表达降低的蛋白和25个表达升高的蛋白,这些蛋白涉及光合作用、二氧化碳固定、糖酵解、蛋白质合成及非正常折叠蛋白降解、细胞分裂和物质运输,以及自由基清除等途径。应用液氮研磨和TCA-丙酮沉淀法可快速高效地提取栅藻Scenedesmus sp.蛋白质,氨氮毒害作用涉及光合作用、脂肪酸合成、非正常折叠蛋白降解等多种代谢途径。     

The effect of illumination on the pigment composition of the ζ-carotenic mutant,PG1, of Scenedesmus obliquus

Dark grown Scenedesmus obliquus strain PG1 accumulated -carotene and phytoene as its major carotenoids. On illumination of dark-grown cultures in air/CO₂, cyclic carotenes and xanthophylls were formed, apparently at the expense of the accumulated phytoene and -carotene. This interconversion of carotenoids was accompanied by chlorophyll synthesis. In an atmosphere of nitrogen/CO₂ the light-induced changes occurred more slowly and in nitrogen alone the changes were incomplete. No massive production of cyclic carotenes from the accumulated -carotene was observed in cultures illuminated under anaerobic conditions.     

M?ssbauer effect in Scenedesmus and spinach ferredoxins. The mechanism of electron transfer in plant-type iron–sulphur proteins

1. The Mössbauer spectra of Scenedesmus ferredoxin enriched in ⁵⁷Fe were measured and found to be identical with those of two other plant-type ferredoxins (from spinach and Euglena) that had been previously measured. Better resolved Mössbauer spectra of spinach ferredoxin are also reported from protein enriched in ⁵⁷Fe. All these iron–sulphur proteins are known to contain two iron atoms in a molecule that takes up one electron on reduction. 2. The Mössbauer spectra at 195°K have electric hyperfine structure only and show that on reduction the electron goes to one of the iron atoms, the other appearing to remain unchanged. 3. In the oxidized state, both iron atoms are in a similar chemical state, which appears from the chemical shift and quadrupole splitting to be high-spin Fe³⁺, but they are in slightly different environments. In the reduced state the iron atoms are different and the molecule appears to contain one high-spin Fe²⁺ and one high-spin Fe³⁺ atom. 4. At lower temperatures (77 and 4.2°K) the spectra of both iron atoms in the reduced proteins show magnetic hyperfine structure which suggests that the iron in the oxidized state also has unpaired electrons. This provides experimental evidence for earlier suggestions that in the oxidized state there is antiferromagnetic exchange coupling, which would result in a low value for the magnetic susceptibility. 5. In a small magnetic field the spectrum of the reduced ferredoxin shows a Zeeman splitting with hyperfine field (H_n) of 180kG at the nuclei. On application of a strong magnetic field H the spectrum splits into two spectra with effective fields H_n±H, thus confirming the presence of the two antiferromagnetically coupled iron atoms. 6. These results are in agreement with the model proposed by Gibson, Hall, Thornley & Whatley (1966); in the oxidized state there are two Fe³⁺ atoms (high spin) antiferromagnetically coupled and on reduction of the ferredoxin by one electron one of the ferric atoms becomes Fe²⁺ (high spin).