Enhancement of plant growth regulators production from microalgae cultivated in treated sewage wastewater (TSW)
提升於處理後污水(TSW)培養之微藻生產植物生長調節劑的能力

Abstract  摘要

The aim of this work is to develop an efficient method for detection and evaluation of the plant growth regulators produced from cyanobacteria species (Anabaena oryzae and Nostoc muscorum) cultivated on BG11₀, and Chlorophyta alga (Chlorella vulgaris) cultivated on BG11 in addition to the cultivation of these strains on treated sewage wastewater (TSW) combined with control media (BG11 and BG11₀) at different concentrations (100, 75 and 50%). Bioassays were performed on Wheat coleoptile length and Cucumber cotyledons fresh weight for indole acetic acid (IAA) and Benzyl adenine (BA) detection. In addition, application experiments of IAA and BA presence in algal extract were applied on tomato plantlets and soybean callus. The obtained results of A. oryzae and N. muscorum extracts (grown on BG11₀ and 100% sewage media) with optimum conc. of IAA and BA showed moderate shoot length and leaves number as well as high root initiation of tomato explant compared to control. While dimethyl sulfoxide (DMSO), IAA conc. as well as IAA + BA conc. showed no effect on branching and leaf expansion. The results of C. vulgaris (grown on BG11) also revealed that the shoot had high leaves number and greatest root initiation, without branching and leaf expansion. On the other hand, 100% TSW had a moderate shoot, leaves number and high root initiation. Extracts of A. oryzae and N. muscorum (grown on BG11₀) induced 1.5-fold increase in soybean callus fresh weight, while the growth on 100% TSW was shown to be less effective. Moreover, extract of C. vulgaris (grown on BG11) induced a moderate effect, while its growth on 100% TSW was shown to be less effective in soybean callus fresh weight increment.
本研究旨在開發一種高效方法，用於檢測和評估由藍藻物種（水稻魚腥藻和苔蘚念珠藻）在 BG11 ₀ 培養基上，以及綠藻門藻類（普通小球藻）在 BG11 培養基上產生的植物生長調節劑，同時將這些菌株培養於不同濃度（100%、75%和 50%）的處理後污水（TSW）與對照培養基（BG11 和 BG11 ₀ ）混合的環境中。針對吲哚乙酸（IAA）和苄基腺嘌呤（BA）的檢測，分別以小麥胚芽鞘長度和黃瓜子葉鮮重進行了生物測定。此外，還將藻類提取物中存在的 IAA 和 BA 應用於番茄幼苗和大豆癒傷組織進行了應用實驗。結果顯示，與對照組相比，A. oryzae 和 N. muscorum 提取物（在 BG11 ₀ 和 100%污水培養基中生長）含有最適濃度的 IAA 和 BA 時，番茄外植體表現出中等程度的芽長和葉片數量，以及較高的根起始率。而二甲基亞碸（DMSO）、IAA 濃度及 IAA+BA 濃度對分枝和葉片擴展無顯著影響。C. vulgaris 的結果... 在 BG11 培養基上生長的尋常小球藻（Chlorella vulgaris）顯示出枝條具有高葉片數量和最強的根起始能力，但無分枝和葉片擴展。另一方面，100% TSW（某種培養基）的枝條表現中等，葉片數量適中且根起始能力較高。米麴黴（Aspergillus oryzae）和苔蘚念珠藻（Nostoc muscorum）（在 BG11 ₀ 培養基上生長）的提取物誘導大豆癒傷組織鮮重增加了 1.5 倍，而在 100% TSW 上生長的效果較差。此外，尋常小球藻（在 BG11 上生長）的提取物產生了中等效果，而在 100% TSW 上生長對大豆癒傷組織鮮重增加的影響較小。

Introduction  引言

Algal cultivation on wastewater could be potentially useful to reduce or even eliminate nutrients and heavy metals in the wastes. This system could use the wastewater instead of being pumped into the ocean and contaminate its water every day. The harvested algal biomass has many potential uses, which include bio-fuel, fish feed and ethanol production. The algae can help the elimination of harmful chemicals out of the wastewater and produce clean drinking water. Microalgal cultures on treated wastewater (urban, industrial or agricultural effluents) can provide a tertiary biotreatment coupled with the production of potentially valuable algal biomass, which can be involved or useful for various purposes [1].
在廢水中培養藻類可能對減少甚至消除廢物中的營養物質和重金屬具有潛在的益處。此系統可利用廢水，而非每天將其泵入海洋並污染海水。收穫的藻類生物質具有多種潛在用途，包括生物燃料、魚飼料和乙醇生產。藻類有助於去除廢水中的有害化學物質，並生產清潔的飲用水。在處理過的廢水（城市、工業或農業廢水）上進行的微藻培養，可以提供三級生物處理，同時生產出可能具有價值的藻類生物質，這些生物質可參與或適用於多種用途[1]。

Several bioactive compounds are produced by cyanobacteria and microalgae have been discovered by screening programs [2]. Many of these chemicals have a diverse range of biological activities and chemical structures, which affect many physiological and biochemical processes within the cell. Such chemicals are thought to be related to the regulation and succession of algal and bacterial populations. These chemicals are expected to be synthesized under stress conditions, low algal growth rate and released at appropriate concentration to be effective.
通過篩選計劃，已發現由藍綠菌和微藻產生的多種生物活性化合物[2]。這些化學物質中的許多具有多樣的生物活性和化學結構，影響細胞內許多生理和生化過程。這類化學物質被認為與藻類和細菌群落的調節及演替有關。預計這些化學物質會在壓力條件下、藻類生長率低時合成，並以適當濃度釋放以發揮效果。

In 1960-1970s an active search for phytohormones in various algal taxa was performed. During this period many compounds with hormonal activity were detected from Chlorophyta, Phaeophyta and Rhodophyta. The most interesting of them are IAA, GA₃, IPA and lunularic acid. Algae produce plant growth regulators [PGRs], similar to those of higher plants. Cyanobacteria synthesized various amounts of auxin in the presence of different concentrations of its precursor L-Tryptophan [3].
在 1960 至 1970 年代，對各種藻類分類群中植物激素的積極搜索被進行。在此期間，從綠藻門、褐藻門和紅藻門中檢測到許多具有激素活性的化合物。其中最引人注目的是 IAA、GA ₃ 、IPA 和月桂酸。藻類產生的植物生長調節劑[PGRs]與高等植物的相似。藍綠菌在其前體 L-色氨酸不同濃度存在下，合成了不同量的生長素[3]。

Many studies focused on the importance of micro-and macro-algal filtrates and extracts either added to the soil or mixed with the tissue culture media or even applied as foliar spray. The effect of seaweed extracts made from the brown algae Durvillaea potatorum and Ascophyllum nodosum on tomato plant and soil was investigated [4,5,6]. They showed a pronounced beneficial effect not only on soil but it can also enhance the morphological, physiological and biochemical parameters of different crop plants and vegetables [7,8,9]. Seaweed extracts contained growth promoting substances as phytohormones and other bio-stimulants like amino acids, vitamins, macro-and micronutrients. These biostimulants may induced an enhancement effect to plant growth [10] by facilitating water uptake, root and shoot growth and tolerance to stresses on all plant developmental stages (as germination of seeds, growth of plants seedlings till plant harvest). Lower concentrations were found to be more effective either applied for seed germination or seedling growth [11, 12].
許多研究聚焦於微藻和大型藻類過濾液及萃取物的重要性，無論是添加至土壤、混合於組織培養基質，或是作為葉面噴施。由褐藻 Durvillaea potatorum 和 Ascophyllum nodosum 製成的海藻萃取物對番茄植株及土壤的影響已被探討[4,5,6]。這些研究顯示，海藻萃取物不僅對土壤有顯著的益處，還能提升不同作物與蔬菜的形態、生理及生化參數[7,8,9]。海藻萃取物含有如植物激素等促進生長的物質，以及其他生物刺激素，如胺基酸、維生素、巨量與微量營養素。這些生物刺激素可能透過促進水分吸收、根莖生長及增強植物各發育階段（從種子發芽、幼苗生長至收成）對逆境的耐受性，從而誘導植物生長的正向效應[10]。研究發現，較低濃度無論應用於種子發芽或幼苗生長，效果更為顯著[11,12]。

Plant growth regulators are found to play a very important role in plant tissue culture (PTC). They are vital for many growth phases. Also, they help in studying the morphological, biochemical, cytological and genetical events of the plants [13]. Auxins and cytokinins are the most widely applied PGRs in plant tissue culture and they are usually added simultaneously. The ratio of auxin to cytokinin control the type of culture to which the explant will develop. Application of higher auxin concentrations stimulates root formation, whereas higher cytokinin regenerates shoot while, intermediate ratio favors callus production. All these events depend on the internal amounts of hormones inside the explants. Since its discovery, PTC had significant and valuable impacts in agricultural sciences and constitutes an essential tool in the advancement of modern agriculture [14].
植物生長調節劑被發現在植物組織培養（PTC）中扮演著非常重要的角色。它們對許多生長階段至關重要。此外，它們有助於研究植物的形態學、生物化學、細胞學和遺傳學事件[13]。生長素和細胞分裂素是植物組織培養中最廣泛應用的植物生長調節劑，通常會同時添加。生長素與細胞分裂素的比例控制著外植體將發展成的培養類型。應用較高濃度的生長素會刺激根部形成，而較高濃度的細胞分裂素則會再生芽體，而中間比例則有利於癒傷組織的產生。所有這些事件都取決於外植體內部的激素量。自其發現以來，PTC 對農業科學產生了重要且有價值的影響，並成為推動現代農業進步的重要工具[14]。

This research aimed to detect and evaluate the plant growth regulators produced from some microalgal species cultivated on low quality water (secondary treated sewage wastewater) and confirm their presence and activity using bioassays and tissue culture application techniques.
本研究旨在檢測並評估由某些微藻物種在低品質水（次級處理後的污水）中培養所產生的植物生長調節劑，並透過生物測定與組織培養應用技術確認其存在與活性。

Materials and methods  材料與方法

Algal samples  藻類樣本

Algal species and culture conditions
藻類物種與培養條件

Two cyanobacterial species (Anabaena oryzae and Nostoc muscorum) were obtained from the Microbiology Department, Soils, Water and Environment Res. Inst. (SWERI), Agric. Res., Center (ARC) and one green alga species (Chlorella vulgaris.) was provided from Dr. Sanaa M. Shanab culture collection, Department of Botany and Microbiology, Faculty of Science, Cairo University, 12,613 Giza, Egypt.
兩種藍綠藻物種（Anabaena oryzae 與 Nostoc muscorum）取自埃及吉薩 12,613 號開羅大學理學院植物學與微生物學系、農業研究中心(ARC)所屬土壤、水與環境研究所(SWERI)微生物部門；另有一種綠藻物種（Chlorella vulgaris）由開羅大學理學院植物學與微生物學系 Sanaa M. Shanab 博士之培養庫提供。

The cyanobacteria species were cultured and maintained on liquid BG11₀ free from nitrogen [15] for cyanobacteria and BG11 media [16] for the green alga. Cultures were incubated in the incubator under continuous aeration (1.25 l/min), 16: 8 h light and dark cycle and light intensity of 40 µE/m²/s at 25 ± 1 °C for 30 days.
藍藻物種於不含氮的液體 BG11 培養基[15]中培養並維持，綠藻則使用 BG11 培養基[16]。培養物在培養箱中連續通氣（1.25 升/分鐘）、16:8 小時光暗循環及 40 µE/m²/s 的光照強度下，於 25±1°C 的環境中孵育 30 天。

Wastewater sources  廢水來源

Sewage Wastewater (SWW, Urban) was obtained from Zenain wastewater station, Al-Eshreen, Giza, Egypt.
生活污水（SWW，城市）採集自埃及吉薩省 Al-Eshreen 的 Zenain 污水處理站。

Wastewater analysis  廢水分析

Chemical indicators and parameters of treated wastewater and BG11 media were analyzed according to APHA [17] as shown in Table 1.
根據 APHA [17] 分析處理後廢水與 BG11 培養基的化學指標與參數，如表 1 所示。

Table 1 Chemical characteristics of the treated sewage wastewater (TSW) used for cultivation of microalgae compared with the composition of BG11 medium
表 1 用於微藻培養的處理後污水（TSW）與 BG11 培養基成分的化學特性比較

Treatment of sewage wastewater
污水處理

The SWW (sewage wastewater) was sterilized by microfiber filter (0.22 µm) to get rid of large particles and bacteria. The wastewater was then termed treated sewage wastewater (TSW). It was applied separately (as 100% TSW) and in combination with BG11₀ or BG11 (in 75, 50%) to study their impacts on algal strains [18]. The BG11₀ or BG11 media were used as control media the (standard synthetic media). The algal strains were grown in 500 ml Erlenmeyer flasks to which 10% algal inocula were applied. Each experiment was conducted in triplicates and cultures were incubated in an illuminated incubator with the previously mentioned conditions.
污水（SWW）通過微纖維過濾器（0.22 µm）進行滅菌處理，以去除大顆粒物和細菌。處理後的污水被稱為處理過的污水（TSW）。該污水被單獨使用（作為 100% TSW）或與 BG11 ₀ 或 BG11（以 75%、50%的比例）混合使用，以研究其對藻類菌株的影響[18]。BG11 ₀ 或 BG11 培養基被用作對照培養基（標準合成培養基）。藻類菌株在 500 毫升錐形瓶中培養，接種 10%的藻類接種物。每項實驗均進行三次重複，培養物在具有前述條件的照明培養箱中培養。

PGRs Extraction  植物生長調節劑提取

The harvested algal cells were dried to powder and extracted by 96% methanol according to the method described in El Akabawy et al. [19].
將收穫的藻類細胞乾燥成粉末，並按照 El Akabawy 等人[19]所述方法，用 96%甲醇進行萃取。

The algal cells were harvested in each experiment at the end of the exponential phase, centrifuged, oven dried (50 °C for 24 h), and extracted overnight in 96% methanol. Then, the methanolic fraction was filtrated, and the residual pellets were re-extracted 3 times with 40% (10 ml) cold methanol. The combined methanol extracts were evaporated in the dark at room temperature. The residual aqueous solution was adjusted to pH 2.6 residual aqueous solutiontimes by absolute ethyl acetate (50 ml/each extract). In addition, the ethyl acetate fraction was separated and dried over anhydrous MgSO4. Finally, the residue was dissolved in 4 ml of absolute methanol.
在每項實驗結束時，於指數生長期末收穫藻類細胞，經離心後於烘箱中乾燥（50°C，24 小時），並以 96%甲醇進行過夜萃取。接著，將甲醇萃取液過濾，殘留的沉澱物再以 40%（每次 10 毫升）冷甲醇重複萃取 3 次。合併的甲醇萃取物於室溫避光條件下蒸發。殘留的水溶液調整至 pH 2.6 後，以純乙酸乙酯（每次 50 毫升）進行萃取。此外，乙酸乙酯層分離後以無水硫酸鎂乾燥。最終，殘留物溶解於 4 毫升純甲醇中。

Experimental research and field studies on plants
植物的實驗研究與田間調查

All Experimental research and field studies on plants, including the collection of plant material (including seeds), comply with relevant institutional, national, and international guidelines and legislation.
所有關於植物的實驗研究與田間調查，包括植物材料（含種子）的採集，均遵守相關機構、國家及國際的指導方針與法規。

Response of the wheat coleoptile section elongation to auxins
小麥胚芽鞘片段對生長素的伸長反應

This method was applied according to Nitsh and Nitsh [20], where: Wheat grains (Triticum aestivum L. Giza 171 was obtained from Agriculture Research Center (ARC), Giza, Egypt) were soaked in distilled water for two hours. The soaked grains were grown in darkness at 25 C for 48 h under green–safe light, then, 6 mm sections below the tips were cutten and10 coleoptile sections of 6 mm length were incubated (in darkness at 25 C for 24 h.) in 2.5 ml sucrose buffer pH 5 with 2.5 ml of algal crude extract of tested samples and 2.5 ml of different IAA conc. (10^–8-10⁻³ M) for dose response curve. Elongation of wheat coleoptile was determined in mm.
該方法依據 Nitsh 與 Nitsh[20]所述進行應用，具體步驟如下：小麥籽粒（品種為 Triticum aestivum L. Giza 171，購自埃及吉薩農業研究中心(ARC)）先以蒸餾水浸泡兩小時。浸泡後的籽粒於 25°C 黑暗環境中，在綠色安全燈光下培養 48 小時，隨後切取尖端下方 6 毫米區段，並將 10 段 6 毫米長的胚芽鞘置於 2.5 毫升 pH 值 5 的蔗糖緩衝液中（含 2.5 毫升待測樣品的藻類粗提物及 2.5 毫升不同濃度 IAA 溶液（10 ^–8 至 10 ⁻³ M，用於劑量反應曲線）），於 25°C 黑暗條件下培養 24 小時。小麥胚芽鞘的伸長量以毫米為單位進行測定。

Cucumber cotyledon fresh weight for cytokinin’s bioassay
黃瓜子葉鮮重用於細胞分裂素的生物測定

Cucumber seeds (Cucumis sativus L. picoline F1-hybrid) were purchased from Germany supermarket) and was kindly identified in the Department of Botany and Microbiology, Faculty of Science, Cairo University. Seeds were germinated in darkness for 4 days at room temperature (25 °C) on moist filter paper in 5-cm petri dishes. Cotyledons were excised excluding petiole tissues and four cotyledons were placed in each petri-dishes after determining their fresh weights. The cotyledons were placed with their ad axial sides down on the paper. They were incubated in the incubator at 25 ± 2 °C and 12/12 h light–dark photoperiods. Three mL of crude extracts of each tested alga was applied to each petri dish at BA concentrations 5, 10, 25, 50 and 100 ppm which were used for dose response curve. Cotyledon growth was measured by determining the increase in fresh weights (in grams) as reported by Letham [21].
黃瓜種子（Cucumis sativus L. picoline F1 雜交種）購自德國超市，並由開羅大學理學院植物學與微生物學系協助鑑定。種子在室溫（25°C）下於 5 公分培養皿中的濕潤濾紙上暗處發芽 4 天。去除葉柄組織後切取子葉，測定鮮重後每培養皿放置四片子葉。子葉以近軸面朝下置於濾紙上，於 25±2°C 培養箱中進行 12/12 小時光暗週期培養。每培養皿添加 3 毫升測試藻類粗萃取物，並使用 5、10、25、50 及 100 ppm 濃度的 BA 進行劑量反應曲線測試。子葉生長量依 Letham[21]所述方法以鮮重增加克數測定。

HPLC conditions  高效液相層析條件

The standard hormonal samples (were purchased from Sigma-Aldrich) and the algal methanol extracts were analyzed by high performance liquid chromatography (HPLC) with the same conditions published in El Akbawy et al. [19]. The amount of IAA and BA in the algae grown on BG11₀ or BG11 (alone and in combinations) with TSW were estimated from the dose growth curves and the HPLC analysis of algal hormones.
標準激素樣品（購自 Sigma-Aldrich）與藻類甲醇提取物，均依照 El Akbawy 等人[19]發表的相同條件，以高效液相層析法（HPLC）進行分析。生長於 BG11 ₀ 或 BG11（單獨及與 TSW 組合）培養基上的藻類，其 IAA 與 BA 含量是根據劑量生長曲線及藻類激素的 HPLC 分析結果估算而得。

Application experiments  應用實驗

A. oryzae, N. muscorum and C. vulgaris grown on different media were selected for application experiments to confirm the presence of IAA and BA by HPLC analyses of algal extracts.
選用在不同培養基上生長的米麴黴、念珠藻和普通小球藻進行應用實驗，通過高效液相色譜（HPLC）分析藻類提取物以確認吲哚乙酸（IAA）和苄基腺嘌呤（BA）的存在。

Soybean callus and Tomato plantlets for Benzyl adenine (BA) and Indol aceticacid (IAA) detection
用於檢測苄基腺嘌呤（BA）和吲哚乙酸（IAA）的大豆癒傷組織與番茄幼苗

The applied methods were developed by Miller [22] and Ran et al. [23].
所採用的方法由 Miller[22]與 Ran 等人[23]開發。

Seed sterilization  種子滅菌處理

The seeds of soybean (Glycine max L. cv. Giza 111) was obtained from Agriculture.
大豆（學名：Glycine max L. 品種：Giza 111）種子取自農業研究中心。

Research Center (NRC), Giza, Egypt) and tomato (Lycopersicon esculentus L. cv. moneymaker) was purchased from USA). The plant seeds were kindly identified in the Department of Botany and Microbiology, Faculty of Science, Cairo University. Seeds sterilization was performed according to the method of Elakbawy et al. [19].
研究來源為埃及吉薩的國家研究中心（NRC），而番茄（學名：Lycopersicon esculentum L. 品種：moneymaker）種子則購自美國。植物種子經開羅大學理學院植物學與微生物學系鑑定。種子滅菌程序依照 Elakbawy 等人[19]所述方法進行。

Seed’s germination  種子的發芽

All seeds (soybean and tomato) were germinated separately in vitro on basal Murashige and Skoog medium (MS) [24]. To which was added 30 g/L sucrose and 100 mg/L myo-inositol and solidified with 0.8% agar. All jars were incubated in growth room with constant temperature (25 ± 0.2ºC) and photoperiod (16 h dark / 8 h light) for7 days.
所有種子（大豆與番茄）分別於基礎穆拉希吉與斯庫培養基（MS）[24]上進行體外發芽。培養基中添加了 30 克/升蔗糖與 100 毫克/升肌醇，並以 0.8%瓊脂固化。所有培養瓶置於恆溫（25±0.2℃）與光週期（16 小時黑暗/8 小時光照）的生長室中培養 7 天。

Seedling of soybean  大豆幼苗

Aseptically growing soybean seedlings of 7 day-old were used as a source of cotyledon explants for callus induction.
以無菌培養 7 日齡的大豆幼苗作為子葉外植體來源，用於誘導癒傷組織形成。

Soybean callus induction  大豆癒傷組織誘導

Soybean callus cultures of were initiated by placing the excised sterile cotyledon-explants (2–3 mm) thickness of 7 day-old. The aseptically grown seedlings were placed in 200 ml screw-capped glass jars containing 25 ml callus induction media. Callus induction medium contained MS supplemented with 0.5 mg/L BA, 30 g/L sucrose, 100 mg/L myo-inositol and 0.8% agar. Five explants per jar (triplicates) were cultured. All cultures were cultured in the dark at constant temperature (25 ± 2 C) for 2 weeks.
大豆癒傷組織培養的起始步驟是將無菌切除的子葉外植體（2-3 毫米厚）取自 7 天大的幼苗。這些在無菌條件下生長的幼苗被置於含有 25 毫升癒傷組織誘導培養基的 200 毫升螺旋蓋玻璃瓶中。癒傷組織誘導培養基包含 MS 基礎培養液，添加了 0.5 毫克/升的 BA、30 克/升的蔗糖、100 毫克/升的肌醇以及 0.8%的瓊脂。每瓶培養五個外植體（三重複）。所有培養物在恆溫（25±2°C）黑暗條件下培養 2 週。

Experiment on soybean callus
大豆癒傷組織實驗

Three pieces of soybean calli were transferred to each jar containing 25 ml of basal MS medium solidified with 0.8% agar as control. Treatments were carried out using different concentration of BA (10, 25 and 50 ppm), crude extract of the algae grown on BG-11_0, BG11 and TSW. Each treatment was represented by 3 jars (as triplicates). The jars were maintained for 28 days at 25 ± 0.2ºC in darkness. Data were determined as fresh mass of calli per jar.
將三塊大豆癒傷組織轉移至每個含有 25 毫升基礎 MS 培養基（以 0.8%瓊脂固化）的玻璃瓶中作為對照組。處理組則使用不同濃度的 BA（10、25 和 50 ppm）、在 BG-11 _0, BG11 和 TSW 上生長的藻類粗提取物進行處理。每個處理組由 3 個玻璃瓶（三重複）代表。這些玻璃瓶在 25±0.2°C 的黑暗條件下維持 28 天。數據以每瓶癒傷組織的鮮重計量。

Seedling of tomato  番茄幼苗

Aseptically grown tomato seedlings (14 day-old) were applied as a source of plantlets.
以無菌培育的番茄幼苗（14 日齡）作為植株來源。

Experiment of tomato seedlings
番茄幼苗實驗

Apical buds of tomato seedlings were excised and transferred to MS media with different additives as described in Elakbawy et al. [19].
切除番茄幼苗的頂芽並轉移至添加不同物質的 MS 培養基中，具體方法如 Elakbawy 等人[19]所述。

Statistical Analysis  統計分析

Data were subjected to an analysis of variance, and the means were compared using 214 the Least Significant Difference (LSD) test at the 0.05 levels (p ≤ 0.05), followed the method described by Snedecor and Cochran [25].
數據進行了變異數分析，並按照 Snedecor 與 Cochran[25]所述方法，在 0.05 顯著水準(p ≤ 0.05)下使用最小顯著差異法(LSD)進行均值比較。

Results and discussion  結果與討論

Growth rate with different treatments
不同處理下的生長速率

The growth rate was expressed as dried mass (mg per 10 ml sample) then evaluated as dry weight (gm/L) and optical density (OD) at 550 nm for cyanobacteria and 660 nm for Chlorophyta. The samples were collected along 30 days with 5 days interval.
生長速率以乾重表示（每 10 毫升樣品的毫克數），隨後評估為乾重（克/升）及在 550 奈米波長下測量的藍綠菌光密度(OD)與 660 奈米波長下測量的綠藻光密度。樣品採集間隔為 5 天，持續 30 天。

Figure 1A showed the growth rates of all Chlorella vulgaris treatments as with the compared control ones. It illustrated that the growth rates have short lag phases (at day 5), then various length of log phases in addition to short decline phases. In case of control medium (BG11 and TSW treatments), the growth rates expressed gradual increment starting from day 1 of cultivation until they reached the optimum biomass productivity then the growth declined. The day of the maximum productivity differ from treatment to another. C. vulgaris treated with control medium BG11 reached maximum growth rates (900 mg/L) at day 25 of cultivation, which is twice the value attained with 50% TSW at day 50 of cultivation. In addition, treating C. vulgaris with control medium (BG11) expressed absolutely high productivity over other treatments even in the decline phase (715 mg/L) at day 30 of cultivation. However, C. vulgaris demonstrated lower growth rates recording 356 and 373 mg/L, respectively at day 50 of cultivation, when treated with 100% and 75% TSW as shown in Table 2. Similarly, optical density method expressed the same trend of C. vulgaris treated with control medium BG11 and the productivity stills high compared to the values of other treatments.
圖 1A 顯示了所有普通小球藻處理組與對照組的生長速率。圖中表明，生長速率在初期有短暫的延滯期（第 5 天），隨後是不同長度的對數期以及短暫的下降期。在對照培養基（BG11 和 TSW 處理）的情況下，生長速率從培養的第 1 天開始逐漸增加，直至達到最佳生物量生產力後開始下降。達到最大生產力的天數因處理而異。使用對照培養基 BG11 處理的普通小球藻在培養的第 25 天達到最大生長速率（900 mg/L），這一數值是 50% TSW 處理在第 50 天達到值的兩倍。此外，即使在下降期（第 30 天培養時為 715 mg/L），使用對照培養基（BG11）處理的普通小球藻也表現出絕對高於其他處理的生產力。然而，如表 2 所示，當使用 100%和 75% TSW 處理時，普通小球藻在培養的第 50 天分別記錄到較低的生長速率，為 356 和 373 mg/L。同樣地，光密度法也表現出普通小球藻相同的趨勢。 使用對照培養基 BG11 處理的尋常小球藻，其生產力仍高於其他處理組的數值。

Fig 1  圖 1

Growth rate by O.D550nm, O.D660nm and Dry wt. ( as g/L) of a) Chlorella vulgaris b) Anabaena oryzae c) Nostoc muscorum cultivated on different conc. of treated sewage wastewater (TSW) in combination with BG11 and BG110
通過 O.D550nm、O.D660nm 及乾重（單位：g/L）測量的生長率：a) 尋常小球藻 b) 稻米魚腥藻 c) 苔蘚念珠藻在不同濃度處理過的污水廢水(TSW)與 BG11 及 BG110 混合培養下的表現

Table 2 Maximum biomass productivity (as mg/L) of algal species cultivated on control media (BG 11 and BG11₀) and treated sewage wastewater (TSW)
表 2 藻類物種在對照培養基（BG 11 及 BG11 ₀ ）與處理後污水（TSW）中培養的最大生物量生產力（以 mg/L 計）

The growth rates of all Anabaena oryzae treatments and control medium (BG11₀) appeared with 3 phases (short lag phase, log phase and short decline phase), as shown in Fig. 1B. It was clearly noticed that the growth rate of control medium (BG11₀) and treatments rise continuously from the first cultivation day until they get the maximum biomass productivity (differ according to treatments) then growth was diminished. Optimum growth rate of A. oryzae cultured on control medium (at day 20 of cultivation) represented about 0.67 of the value recorded at day 25 of cultivation when treated with 100% TSW (600 mg/L). Also, treating A. oryzae with 100% TSW (at day 30) and control medium (at day 25) expressed maximum productivity (400 mg/L) compared to those of other treatments as shown in Table 2. However, low growth rates expressed 50 and 300 mg/L at day 15 of cultivation, when treated with 50% and 75% TSW, respectively which was lower than the optimum value recorded by control medium.
如圖 1B 所示，所有 Anabaena oryzae 處理組及對照培養基（BG11 ₀ ）的生長速率均呈現三個階段（短暫延滯期、對數期及短暫衰退期）。可明顯觀察到，對照培養基（BG11 ₀ ）與各處理組的生長速率自培養首日起持續上升，直至達到最大生物量生產力（數值因處理而異），隨後生長減緩。在對照培養基上培養的 A. oryzae 最佳生長速率（培養第 20 天）約為使用 100% TSW 處理時（600 mg/L，培養第 25 天）記錄值的 0.67 倍。此外，如表 2 所示，以 100% TSW 處理（第 30 天）及對照培養基（第 25 天）培養的 A. oryzae 表現出最大生產力（400 mg/L），優於其他處理組。然而，當使用 50%及 75% TSW 處理時，於培養第 15 天記錄到的低生長速率分別為 50 mg/L 與 300 mg/L，低於對照培養基所記錄的最佳值。

The growth rates of all Nostoc muscorum treatments and control BG11₀ were manifested in Fig. 1C. The growth rate of control medium (BG11₀) and treatments showed similar trends but vary with the days of lag, maximum productivity and decline. Maximum productivity of N. muscorum as shown in Table 2 reached about 350 mg/L at day 20 of cultivation when treated with control medium (BG11₀). However, treatment with 50% TSW expressed lowest growth rate recording 109 mg/L (at day 15 of cultivation), which was about 1/3 the value recorded with control medium (350 mg/L) at day 20 of growth. Treating N. muscorum with 75% TSW and 100%TSW represented about 72 (at day 30) and 78% (at day 25), respectively of the optimum productivity of that expressed with control medium.
所有 Nostoc muscorum 處理組及對照組 BG11 ₀ 的生長速率如圖 1C 所示。對照培養基（BG11 ₀ ）與處理組的生長趨勢相似，但在延滯期天數、最大生產力及衰退階段有所差異。如表 2 所示，當使用對照培養基（BG11 ₀ ）處理時，N. muscorum 的最大生產力於培養第 20 天達到約 350 mg/L。然而，使用 50% TSW 處理的組別表現出最低生長速率，記錄值為 109 mg/L（培養第 15 天），約為對照培養基（350 mg/L）於生長第 20 天記錄值的 1/3。以 75% TSW 及 100% TSW 處理 N. muscorum，其生產力分別約為對照培養基最佳生產力的 72%（第 30 天）與 78%（第 25 天）。

The results revealed that maximum biomass productivity was enhanced using 100% TSW in case of cyanobacterial species (A. oryza & N. muscorum) and using 50% TSW for green C. vulgaris than the control one (BG11 or BG11₀). These may be due to the richness of TSW with macro and micro-elements in addition to IAA, BA and NPK compared to the utilized synthetic media (BG11 and BG11₀).
結果顯示，對於藍藻物種（A. oryza 與 N. muscorum），使用 100% TSW 能提升最大生物量生產力；而對於綠藻 C. vulgaris，使用 50% TSW 的效果優於對照組（BG11 或 BG11 ₀ ）。這可能歸因於 TSW 富含大量及微量元素，並含有 IAA、BA 及 NPK，相較於所使用的合成培養基（BG11 與 BG11 ₀ ）更為豐富。

The results were in context with those reported by Wang et al., [18] who mentioned that, some microalgae (as C. vulgaris) could effectively acclimatize on various wastewaters (municipal wastewater treatment plant, MWTP) without lag phase. Similarly, Shalaby et al., [26] described that N. muscorum was adapted well when cultivated on several types of wastewaters (Sewage, Industrial and Agriculture).
這些結果與 Wang 等人[18]所報告的內容相符，他們提到某些微藻（如普通小球藻）能夠有效適應各種廢水（市政污水處理廠，MWTP）而無需滯伏期。同樣地，Shalaby 等人[26]描述說，當培養於多種類型的廢水（生活污水、工業與農業廢水）時，N. muscorum 表現出良好的適應性。

Table 1 recorded the chemical analysis of both TSW and BG11₀ media which overlook the reason that TSW stimulated the higher growth rates of algae. TSW was slightly acidic while BG11₀ medium was neutral. Both TSW and BG11₀ media have equal content of potassium. The total nitrogen amount was approving in TSW medium than of those of BG11₀. Also, TSW possessed high amounts of anions and trace elements (Cu, Zn and Co) necessary in enzymes and biochemical processes. While, the amounts of boron, phosphate, iron and manganese were higher in BG11₀ medium.
表 1 記錄了 TSW 與 BG11 ₀ 培養基的化學分析結果，這解釋了為何 TSW 能刺激藻類更高的生長速率。TSW 呈微酸性，而 BG11 ₀ 培養基則為中性。TSW 與 BG11 ₀ 培養基的鉀含量相當。TSW 培養基中的總氮量較 BG11 ₀ 更為充足。此外，TSW 含有高量的陰離子及對酶與生化過程必需的微量元素（銅、鋅和鈷）。而硼、磷酸鹽、鐵和錳的含量則在 BG11 ₀ 培養基中較高。

Dose response curve of IAA
IAA 的劑量反應曲線

Table 3 recorded the elongation of wheat coleoptile sections after treatment by various IAA concentrations (10^–8- 10^–3 M). The results revealed that the increase in exogenous IAA concentrations caused a gradual enhancement in wheat coleoptile sections elongation up to the conc. 10^–5 M IAA where maximum elongation was recorded (8.9 mm, 70.6%) and compared to that obtained by control (7.7 mm). Elevation of IAA conc. (10^–4, 10^–3 M) showed an inhibitory effect
表 3 記錄了經不同濃度 IAA（10 ^–8 - 10 ^–3 M）處理後小麥胚芽鞘切段的伸長情況。結果顯示，隨著外源 IAA 濃度的增加，小麥胚芽鞘切段的伸長逐漸增強，直至 IAA 濃度達到 10 ^–5 M 時記錄到最大伸長量（8.9 毫米，70.6%），與對照組（7.7 毫米）相比有顯著提升。當 IAA 濃度進一步提高（10 ^–4 、10 ^–3 M）時，則表現出抑制作用。

Table 3 Dose response curve of Indole acetic acid (IAA) using wheat coleoptile section elongation (in mm) All data
表 3 使用小麥胚芽鞘切段伸長（毫米）的吲哚乙酸（IAA）劑量反應曲線 所有數據

The replacement of the exogenous IAA concentration by crude algal extract of A. oryzae cultivated on BG11₀ shown an appreciable positive result with more elongation values than that produced by 10^–4 M.
以培養於 BG11 ₀ 培養基的米麴菌粗藻提取物替代外源 IAA 濃度後，顯示出顯著的正面效果，其促伸長值甚至超過 10 ^–4 M IAA 所產生的效果。

IAA (7.92, 12.9%), while crude extracts of both Chlorella vulgaris (cultivated on BG11) and Nostoc muscurum grown on BG11₀ shown negative result with elongation values which were approximately equal to that produced by 10^–4 M IAA (7.5, -11.8% and 7.6, -5.9%) as recorded in Table 3.
IAA（7.92，12.9%），而培養於 BG11 培養基上的普通小球藻（Chlorella vulgaris）及同樣培養於 BG11 培養基上的念珠藻（Nostoc muscurum）的粗提取物，其伸長值顯示為負結果，約等同於 10 ^–4 M IAA 所產生的伸長值（7.5，-11.8% 和 7.6，-5.9%），如表 3 所記錄。

The induced elongation of wheat coleoptile sections (after the removal of tip rich in auxin), by applying exogenous IAA, could be explained as following: The plant cell wall is rigid and not easy to expand due to its composition of cellulose microfibrils mixed with polysaccharides and embedded in hemicellulose, pectin, proteins and other components which are held together by hydrogen bonding. So the expansion of cell wall need the change of its physical properties, where disruption of the cellulose microfibrils structure and loosening the link with polysaccharides takes place by auxins especially at its optimum conc. Auxins promote acidification of the cell wall(due to acid growth theory), stimulating the plasma membrane in the cytoplasm to pump protons (H⁺) to the cell wall which induce loosening enzymes to loosen the cross links between the cellulose microfibrils, polysaccharides and the hydrogen bonding and so microfibrils displace to slide past each other allowing the cell wall to increase its extensibility and plasticity to expand. Then the bonds are reformed in the new position and microfibrils stimulated inducing irreversible cell wall growth [27].
施加外源 IAA 所誘導的小麥胚芽鞘切段（在去除富含生長素的尖端後）伸長現象，可解釋如下：植物細胞壁因其由纖維素微纖維與多醣混合組成，並嵌入半纖維素、果膠、蛋白質及其他由氫鍵結合的組分中，故具有剛性且不易擴展。因此，細胞壁的擴張需要其物理性質的改變，即生長素（特別是在其最適濃度下）破壞纖維素微纖維結構並鬆解其與多醣間的連結。根據酸生長理論，生長素促進細胞壁酸化，刺激細胞質中的質膜將質子（H+）泵入細胞壁，誘導鬆解酶鬆開纖維素微纖維、多醣及氫鍵間的交叉連結，使微纖維得以相互滑移，從而增加細胞壁的可延伸性與可塑性以利擴張。隨後，鍵結在新位置重新形成，受刺激的微纖維誘導不可逆的細胞壁生長[27]。

Dose response curve of BA
BA 的劑量反應曲線

Table 4 recorded the growth curve of the exogenously added BA to cucumber cotyledons using concentrations 5- 100 ppm.
表 4 記錄了外源添加 BA 濃度在 5-100 ppm 範圍內對黃瓜子葉生長的影響曲線。

Table 4 Dose response curve of Benzyl adenine (BA) using fresh wt. (gm) of cucumber cotyledon
表 4 使用黃瓜子葉鮮重（克）繪製的苯甲基腺嘌呤（BA）劑量反應曲線

The obtained data revealed that, lower concentration of BA (5 and 10 ppm) induced gradual increase in cotyledon fresh weight which reached its maximum weight (0.123 g) at the optimum BA conc. (25 ppm) compared to that of the control plant (0.127 g).
獲得的數據顯示，與對照植物（0.127 克）相比，較低濃度的 BA（5 和 10 ppm）誘導了子葉鮮重的逐漸增加，並在最佳 BA 濃度（25 ppm）時達到最大重量（0.123 克）。

Elevation of BA conc. (50 and 100 ppm) caused a steady constant inhibition of growth (≈0.08 g) compared to those recorded by lower BA conc. (5 and10 ppm).
與較低 BA 濃度（5 和 10 ppm）記錄的數據相比，提高 BA 濃度（50 和 100 ppm）導致生長持續受到抑制（≈0.08 克）。

The replacement of exogenous BA conc. by crude algal extract of A. oryzae (cultivated on BG11₀) was shown to produce 0.006 g of cucumber cotyledon fresh weight with 0.24 ppm. Whereas crud algal extract of N. muscorum cultivated on BG11₀ was shown to produce 0.0072 g of cucumber cotyledon fresh weight induced by 0.6 ppm and C. vulgaris crud extract (cultivated on BG11) produced 0.0002 g with 0.017 ppm.
以培養於 BG11 ₀ 的米麴菌粗藻提取物替代外源 BA 濃度，顯示在 0.24 ppm 下可產生 0.006 克的黃瓜子葉鮮重。而培養於 BG11 ₀ 的念珠藻粗藻提取物在 0.6 ppm 下誘導產生 0.0072 克的黃瓜子葉鮮重，培養於 BG11 的小球藻粗提取物則在 0.017 ppm 下產生 0.0002 克。

The optimum conc. of IAA and BA in Tables 3 and 4 were used to calculate the hormone conc. in the application experiments using wastewater, BG11 and BG11_o media as well as IAA + BA combinations.
表 3 和表 4 中的 IAA 與 BA 最佳濃度被用於計算應用實驗中的激素濃度，實驗中使用了廢水、BG11 及 BG11 _o 培養基，以及 IAA 與 BA 的組合。

HPLC analysis results  高效液相層析分析結果

Analysis of plant growth regulators IAA and BA in different extracts of the tested algae cultivated in TSW (100, 75 and 50%), BG11 and BG11₀ media were performed using HPLC as shown in Table 5. The analyses showed that, IAA content in Chlorella vulgaris was increased in alga cultivated in 50 and 100% TSW and BG11 combination (0.014 and 0.032 mg/g) when compared with only BG11 medium (0.01 mg/g). While, the other results showed decreased values compared with BG11 medium. BA amount was increased in 100, 75 and 50% TSW and BG11 combination (0.695, 0.005 and 0.033 mg/g). While, TSW 100% recorded the highest concentration of IAA and BA when compared with values obtained by BG11 medium.
如表 5 所示，使用高效液相層析法分析了在 TSW（100%、75%和 50%）、BG11 及 BG11 ₀ 培養基中培養的測試藻類不同提取物中的植物生長調節劑 IAA 和 BA。分析結果顯示，與僅使用 BG11 培養基（0.01 mg/g）相比，小球藻（Chlorella vulgaris）在 50%和 100% TSW 與 BG11 混合培養基中的 IAA 含量增加（分別為 0.014 和 0.032 mg/g）。而其他結果顯示，與 BG11 培養基相比，數值有所下降。BA 的含量在 100%、75%和 50% TSW 與 BG11 混合培養基中增加（分別為 0.695、0.005 和 0.033 mg/g）。其中，100% TSW 記錄到的 IAA 和 BA 濃度最高，高於 BG11 培養基所得數值。

Table 5 HPLC analysis of PGRs (as mg/g) extracted from cyanobacteria species and Chlorophyta alga cultivated on BG11₀ or BG11 (as a control) and TSW media
表 5 從在 BG11 ₀ 或 BG11（作為對照）及 TSW 培養基中培養的藍藻物種和綠藻門藻類提取的植物生長調節劑（單位：mg/g）之高效液相層析分析

The obtained results of different Anabaena oryzae extracts cultivated in TSW and BG11₀ media revealed that, both IAA and BA contents were increased in alga cultivated in BG11₀ and TSW combination media (50, 75 and 100%), when compared with control (BG11₀ medium). Moreover, 100% TSW recorded highest concentration of IAA (0.051 mg/g) when compared with its content when grown on BG11₀ medium (0.003 mg/g). However, algae cultivated in 50% TSW recorded the highest concentration of BA (0.044 mg/g) when compared with those recorded on using BG11₀ medium (0.013 mg/g).
不同來源的魚腥藻提取物在 TSW 與 BG11 ₀ 培養基中的實驗結果顯示，相較於對照組（BG11 ₀ 培養基），在 BG11 ₀ 與 TSW 混合培養基（50%、75%及 100%）中生長的藻類，其 IAA 與 BA 含量均有所提升。此外，100% TSW 培養條件下記錄到的 IAA 濃度最高（0.051 毫克/克），顯著高於 BG11 ₀ 培養基中的含量（0.003 毫克/克）。然而，50% TSW 培養的藻類其 BA 濃度達到峰值（0.044 毫克/克），相較於 BG11 ₀ 培養基中的記錄值（0.013 毫克/克）有顯著差異。

The analysis of various extracts of N. muscorum showed that, IAA content was elevated in alga cultivated in TSW (50, 75 and 100%), BG11₀ combination over that of control. The amount of BA was improved in 100%, 50% TSW, BG11₀ combination media and declined in 75% TSW. However, the highest concentration of IAA and BA was recorded in 100%TSW when compared with BG11₀ medium.
對念珠藻多種提取物的分析表明，相較於對照組，在 TSW（50%、75%及 100%）與 BG11 ₀ 混合培養基中生長的藻類，其 IAA 含量有所增加。BA 的含量則在 100%與 50% TSW 及 BG11 ₀ 混合培養基中提升，但在 75% TSW 中下降。值得注意的是，與 BG11 ₀ 培養基相比，100% TSW 條件下記錄到的 IAA 與 BA 濃度均為最高。

These results showed that the abiotic stress performed by the investigated algae with treated wastewater (rich in macro and micro nutrients as seen in Table 1, could stimulate algal cells to induce various secondary metabolites as self-defense. These obtained results were in accordance with those recorded by Rodríguez-Meizoso et al. [28] who mentioned that when Microalgae face stress and/or extreme natural environmental conditions, they rapidly acclimatize themselves to the new conditions for survival. In this process, they induce new biologically active secondary metabolites which are not synthesized under normal unstressed conditions.
這些結果顯示，研究藻類在處理過的廢水（富含宏量和微量營養素，如表 1 所示）中進行的非生物脅迫，可能刺激藻類細胞誘導各種次級代謝產物作為自我防禦。這些獲得的結果與 Rodríguez-Meizoso 等人[28]記錄的結果一致，他們提到當微藻面臨脅迫和/或極端的自然環境條件時，它們會迅速適應新條件以生存。在此過程中，它們誘導出新的生物活性次級代謝產物，這些代謝產物在正常無脅迫條件下不會合成。

Tomato experiment  番茄實驗

This invitro experiment tested the influence of different auxin (IAA) and cytokinin (BA) concentrations (separately and in combination), in addition to the tested algal optimum conc of these hormones and its crude extracts obtained from cultures grown on various tested media (synthetic and cheap media). MS and DMSO media was used as controls. The impact was focused on the morphological parameters (shoot length, leaves numbers, leaf expansion, roots initiation and branching) of tomato plantlets.
這項體外實驗測試了不同濃度生長素（IAA）和細胞分裂素（BA）（單獨及組合使用）的影響，此外還測試了藻類對這些激素的最佳濃度及其從各種測試培養基（合成培養基和廉價培養基）中生長的培養物中獲得的粗提取物。MS 和 DMSO 培養基被用作對照。影響主要集中在番茄幼苗的形態參數（芽長、葉片數量、葉片擴展、根起始和分枝）上。

Table 6 and Fig. 2 revealed that A. oryzae which showed that, IAA at conc. 1.8 ppm stimulated the greatest shoot length (6.850 ± 1.588) compared to MS and DMSO media, other IAA conc. (0.5 and 2.5 ppm) and IAA + BA conc. This unbranched shoot possessed high leaves number (3.583 ± 0.515) and greatest root initiation. High IAA conc. (2.5 ppm) was highly effective on leaves number and shoot length than the lower IAA conc. (0.5 ppm).
表 6 與圖 2 顯示，米麴黴（A. oryzae）在濃度 1.8 ppm 的 IAA 處理下，相較於 MS 與 DMSO 培養基、其他 IAA 濃度（0.5 與 2.5 ppm）及 IAA+BA 組合濃度，能顯著促進最長芽長（6.850 ± 1.588）。此未分枝芽體具有高葉片數（3.583 ± 0.515）與最佳根系誘發效果。高濃度 IAA（2.5 ppm）對葉片數與芽長的促進效果顯著優於低濃度 IAA（0.5 ppm）。

Table 6 The effect of Anabaena oryzae crude extracts (Grown on BG11₀ and 100% TSW) with optimum conc. of acetic acid (IAA) and Benzyl adenine (BA) (obtained from dose response curve) on Tomato plant
表 6 魚腥藻（Anabaena oryzae）粗萃取物（培養於 BG11 ₀ 與 100% TSW 基質）搭配最適濃度乙酸（IAA）與苄基腺嘌呤（BA）（由劑量反應曲線取得）對番茄植株之影響

Fig 2  圖 2

Growth of tomato explants by syn. BA, IAA and mixtures of IAA + BA conc. as well as algal extracts (grown on various media) with optimum conc. of BA and IAA (from dose response curve) on tomato plant
番茄外植體的生長情況，通過合成 BA、IAA 及其混合濃度（IAA+BA）以及在不同培養基上生長的藻類提取物（配合 BA 和 IAA 的最佳濃度，基於劑量反應曲線）對番茄植株的影響進行評估。

Moderate conc. of exogenously applied hormones (IAA + BA, 1.8 + 25 ppm) enhanced the morphological parameters of tomato explants. Similar results were observed by A. oryzae extract cultivated on BG11₀ supplied with 0.5 IAA + 10 BA ppm. However, higher IAA + BA mixture conc. (2.5 + 50 ppm) expressed lesser stimulatory effects to all morphylogical parameters. A. oryzae extracts (cultured on BG11₀ and 100% TSW media), has no effect on branching and leaf expansion but stimulated high root initiation with moderate shoot length and leaves number compared to control, DMSO, IAA conc. and IAA + BA concentration. Table 6 and Fig. 2 revealed that the crude extract of A. oryzae with optimum conc. of BA expressed moderate shoot length, leaves number and root initiation.
適量外源激素（IAA + BA，1.8 + 25 ppm）的施用顯著提升了番茄外植體的形態學參數。類似結果亦見於在添加 0.5 IAA + 10 BA ppm 的 BG11 ₀ 培養基上培養的米麴菌提取物。然而，更高濃度的 IAA + BA 混合液（2.5 + 50 ppm）對所有形態參數的刺激作用較弱。相較於對照組、DMSO、單獨 IAA 濃度及 IAA+BA 組合濃度，米麴菌提取物（培養於 BG11 ₀ 與 100% TSW 培養基）雖未影響分枝與葉片擴展，但顯著促進了根系發生，同時維持中等程度的芽長與葉片數量。表 6 與圖 2 顯示，含有最佳 BA 濃度的米麴菌粗提物能誘導中等芽長、葉片數及根系發生。

Table 7 and Fig. 2 presented that, indole acetic acid (1.8 ppm) exhibited the greatest shoot length (6.850 ± 1.588), leaves number (3.583 ± 0.515) and root initiation compared to control media, other IAA conc. (0.5 and 2.5 ppm) and the combination IAA + BA concentration. N. muscorum extract (grown on BG11₀ and 100% TSW media) had moderate shoot length and high leaves number of tomato explant Highest root initiation were recorded by utilizing 100% TSW and moderate was observed by BG11₀ medium. The crude extract of N. muscorum supplemented with optimum conc. of BA (grown on BG11₀ and 100% TSW) stimulated moderate shoot length (4.892 ± 0.470, 4.833 ± 0.597) as well as low root initiation and leaves number (3.250 ± 0.754, 3.083 ± 0.669), Table 7 and Fig. 2.
表 7 與圖 2 顯示，相較於對照培養基、其他 IAA 濃度（0.5 與 2.5 ppm）及 IAA+BA 組合濃度，吲哚乙酸（1.8 ppm）表現出最佳芽長（6.850 ± 1.588）、葉片數（3.583 ± 0.515）與根系誘導效果。培養於 BG11 ₀ 與 100% TSW 培養基的 N. muscorum 萃取物對番茄外植體呈現中等芽長與高葉片數。使用 100% TSW 培養基時記錄到最高根系誘導率，而 BG11 ₀ 培養基則表現中等。添加最適 BA 濃度的 N. muscorum 粗萃取物（培養於 BG11 ₀ 與 100% TSW）雖刺激中等芽長（4.892 ± 0.470, 4.833 ± 0.597），但根系誘導率與葉片數（3.250 ± 0.754, 3.083 ± 0.669）較低，詳見表 7 與圖 2。

Table 7 The effect of Nostoc muscorum crude extracts (Grown on BG11₀ and 100% TSW) with optimum conc. of acetic acid (IAA) and benzyl adenine (BA) (obtained from dose response curve) on Tomato plant
表 7 培養於 BG11 ₀ 與 100% TSW 的 Nostoc muscorum 粗萃取物（含最適濃度乙酸 IAA 與苯甲酸腺嘌呤 BA，濃度由劑量反應曲線取得）對番茄植株之影響

The results of C. vulgaris as shown in Table 8 and Fig. 2 revealed that 1.8 ppm indole acetic acid (IAA) reported the formation of greatest shoot length (6.850 ± 1.588, 6.817 ± 0.746) compared to other treatments. The shoot possessed high leaves number (3.583 ± 0.515, 3.833 ± 0.389) and greatest root initiation. However, moderate shoot length (5.050 ± 0.585), leaves number (3.750 ± 0.965) and high root initiation were obtained by 100% TSW. Both lower IAA conc. (0.5 ppm) and higher conc. (2.5 ppm) have less influence on shoot length and leaves number than other treatments. All morphological parameters of tomato explants were stimulated by moderate IAA + BA combination, (1.8 + 2.5 ppm). Similar results were observed by C. vulgaris extract when cultivated on BG11 supplied with 0.5 IAA + 10 BA ppm. In addition, moderate shoot length, leaves number and root initiation were reported by applying the crude extract of C. vulgaris grown on 100% TSW, Table 8 and Fig. 2.
如表 8 及圖 2 所示，普通小球藻（C. vulgaris）的結果顯示，1.8 ppm 的吲哚乙酸（IAA）相較於其他處理，報告了最大的芽長（6.850 ± 1.588，6.817 ± 0.746）形成。該芽具有高葉片數（3.583 ± 0.515，3.833 ± 0.389）及最佳的根起始。然而，100% TSW 則獲得了中等芽長（5.050 ± 0.585）、葉片數（3.750 ± 0.965）及高根起始。較低 IAA 濃度（0.5 ppm）與較高濃度（2.5 ppm）對芽長及葉片數的影響均小於其他處理。番茄外植體的所有形態參數均由中等 IAA + BA 組合（1.8 + 2.5 ppm）刺激。類似結果在普通小球藻提取物於添加 0.5 IAA + 10 BA ppm 的 BG11 培養基上培養時亦被觀察到。此外，施用生長於 100% TSW 上的普通小球藻粗提取物亦報告了中等芽長、葉片數及根起始，詳見表 8 及圖 2。

Table 8 The effect of Chlorella vulgaris crude extracts (Grown on BG11 and 100% TSW) with optimum conc. of acetic acid (IAA) and benzyl adenine (BA) (obtained from dose response curve) on Tomato plant
表 8 普通小球藻（Chlorella vulgaris）粗提取物（生長於 BG11 及 100% TSW 上）與最佳濃度乙酸（IAA）及苯甲基腺嘌呤（BA）（由劑量反應曲線獲得）對番茄植株的影響

Soybean experiment  大豆實驗

Table 9 and Fig. 3 showed the application of cytokinin (BA) bioassay experiment using different concentrations. as well as the effect of crude extracts of the cyanobacterial species A. oryzae and N. muscorum grown on BG11₀ and 100% TSW media and the green alga Chlorella vulgaris which was grown on BG11 and 100 TSW % on fresh weight of soybean callus. Basal MS medium represent the control.
表 9 與圖 3 展示了使用不同濃度的細胞分裂素（BA）生物測定實驗的應用，以及生長於 BG11 ₀ 與 100% TSW 培養基上的藍藻物種 A. oryzae 和 N. muscorum 粗提物，以及生長於 BG11 與 100% TSW 上的綠藻 Chlorella vulgaris 對大豆癒傷組織鮮重的影響。基礎 MS 培養基作為對照組。

Table 9 Effect of crude extracts of algal species on soybean callus fresh weight ( as gm)
表 9 藻類物種粗提物對大豆癒傷組織鮮重的影響（單位：克）

Fig 3  圖 3

Effect of synthetic BA different concentrations as well as algal crude extracts (grown on BG11 and TSW media) on soybean callus fresh weight (g).
合成 BA 不同濃度及藻類粗提物（培養於 BG11 與 TSW 培養基）對大豆癒傷組織鮮重（克）之影響。

The callus weight in control plant was highly decreased (-0.093 g) while the addition of BA in different concentrations. (10, 25 and 50 ppm) induced significant increase in callus weight which reached its maximum at BA concentration 25 ppm (2.8397 g).The obtained results concerning soybean fresh weight, revealed that A. oryzae and N. muscorum extracts (contained BA) after growth on BG11₀ medium (contained 10–25 ppm BA) induced 1.5-fold increase in callus fresh weight, while when growth on 100% TSW medium (contained > 10 ppm BA) was showed to be less effective in fresh weight increment. Concerning, C. vulgaris the results showed that, its extract (contained BA) after growth on BG11 (contained 10–25 ppm BA) induced a moderate effect, but when growth on 100% TSW medium (contained > 10 ppm BA) it was showed to be less effective in fresh weight increment.
對照組植物之癒傷組織重量顯著下降（-0.093 克），而添加不同濃度 BA（10、25 及 50 ppm）則誘導癒傷組織重量顯著增加，並於 BA 濃度 25 ppm 時達到最大值（2.8397 克）。關於大豆鮮重之研究結果顯示，生長於 BG11 ₀ 培養基（含 10-25 ppm BA）之米麴黴與念珠藻提取物（含 BA）可誘導癒傷組織鮮重增加 1.5 倍；然當其生長於 100% TSW 培養基（含>10 ppm BA）時，對鮮重增加之效果較不顯著。至於小球藻之結果表明，其生長於 BG11 培養基（含 10-25 ppm BA）之提取物（含 BA）具有中等效果，但當生長於 100% TSW 培養基（含>10 ppm BA）時，對鮮重增加之效果亦較不顯著。

The analysis of aqueous commercial seaweed filtrate, micro-algal or cyanobacterial extracts in many published researches revealed that, they contained not only different plant growth hormones (auxins, cytokinin’s, and gibberellins), but also polyamines, betaine, brasinosteroids, in addition to micro-, macronutrients and vitamins to which attributed the growth improvement [29,30,31,32]. So, they were termed bio-fertilizers or bio-stimulants and widely used as nontoxic, biodegradable and cheap fertilizers to replace the toxic expensive chemical ones [5, 33].
許多已發表的研究對商業海藻濾液、微藻或藍藻提取物的分析顯示，這些物質不僅含有不同的植物生長激素（如生長素、細胞分裂素和赤黴素），還包含多胺、甜菜鹼、油菜素內酯，以及微量與巨量營養素和維生素，這些成分被認為是促進植物生長的原因[29, 30, 31, 32]。因此，這些提取物被稱為生物肥料或生物刺激素，並廣泛用作無毒、可生物降解且價格低廉的肥料，以取代有毒且昂貴的化學肥料[5, 33]。

Plant growth regulators are universally used for enhancing growth and development of many economic crops and vegetables [9, 11, 31, 32, 34]. They are naturally occurring organic compounds which influence the physiological processes that control cell division, embryogenesis, as well as the sequential growth and differentiation processes that are involved in the course of plant reproduction [31, 35, 36]. The endogenously produced plant hormones (especially IAA) in algal and cyanobacterial filtrates or extracts were correlated with root promotion, cell elongation, division, tissue differentiation and responses to light and gravity [37, 38]. Cytokinin’s are produced by many algae and cyanobacteria and play a vital role in regulating growth and morphogenesis. It induces shoots [39], direct and indirect plant regeneration [40], induce callus irrespective of explants used [9] as well as plant regeneration from callus by changing the applied percentage of IAA and cytokinin (the key hormones).
植物生長調節劑被廣泛用於促進多種經濟作物和蔬菜的生長與發育[9, 11, 31, 32, 34]。這些天然存在的有機化合物能影響控制細胞分裂、胚胎發生，以及在植物繁殖過程中涉及的連續生長與分化過程的生理活動[31, 35, 36]。藻類和藍綠菌濾液或提取物中內源性產生的植物激素（特別是 IAA）與根部促進、細胞伸長、分裂、組織分化以及對光和重力的反應相關[37, 38]。細胞分裂素由許多藻類和藍綠菌產生，在調節生長和形態發生中扮演關鍵角色。它能誘導芽的形成[39]、直接和間接的植物再生[40]、不論使用何種外植體均能誘導癒傷組織[9]，以及通過改變 IAA 和細胞分裂素（關鍵激素）的應用比例來實現從癒傷組織的植物再生。

The obtained results in the application experiment were in agreement with the results obtained by Suresh et al., [34] who treated crop plants (zea mays,sorghum bicolour) with heterocystous cyanobacterial filtrate (0.2–0.5%) which promoted grain seedling growth. Also, using similar bio-stimulants [35, 41] reported an amplification of seed germination percentage and root, shoot lengthening of the germinated plants. Moreover, micro-algal extracts (Chlorella vulgaris, Scenedesmus obliquus, S. quadricauda and Cladophoropsis gerloffi) improved lettuce seedlings quality and quantity [6, 33], sugar beet early stages of growth [36] and tomato [31, 32, 42]. Meanwhile, seaweed extracts and seaweed commercial filtrates were widely applied on canola plant by Hashem et al., [5], where it improved growth, yield and alleviated the harmful effect of salinity stress. It was also applied to Ocimum santicum [43] to Brassica chinensis [8], to water stressed tomato [32] and to crop plants [30]. The stimulatory effect of seaweed filtrates and microalgal extracts were proved to be due to the presence of many growth regulators as auxins, cytokinins, gibberellin, polyamines and betaine in addition to different macro-and micro-nutrients as reported by many investigators [44,45,46,47,48,49].
應用實驗中獲得的結果與 Suresh 等人[34]的研究一致，他們使用異形胞藍藻濾液（0.2–0.5%）處理作物（玉米、高粱），促進了穀物幼苗的生長。同樣地，使用類似的生物刺激劑[35,41]報告了種子發芽率的提高以及發芽植物根部和莖部的延長。此外，微藻提取物（普通小球藻、斜生柵藻、四尾柵藻和 Gerloffi 枝藻）改善了萵苣幼苗的質量和數量[6,33]，甜菜生長初期[36]以及番茄[31,32,42]。同時，海藻提取物和商業海藻濾液被 Hashem 等人[5]廣泛應用於油菜植物，改善了生長、產量並減輕了鹽分脅迫的有害影響。它還被應用於聖羅勒[43]、小白菜[8]、水分脅迫下的番茄[32]以及作物植物[30]。 如多位研究者所報告[44, 45, 46, 47, 48, 49]，海藻濾液與微藻萃取物的刺激效應已被證實源自其中含有多種生長調節物質（如生長素、細胞分裂素、吉貝素、多胺及甜菜鹼）以及不同巨量與微量營養素。

Conclusion  結論

We have successfully detected and determined auxins and cytokinin (IAA and BA) which were endogenous produced phytohormones by cyanobacteria (A. oryzae and N. muscorum) and Chlrophyta (C. vulgaris) species. The extracts of algae grown on different conc. of treated wastewater combined with BG11 or BG11₀ media showed a significant effect on morphological parameters of tomato plantlets (shoot length, branching, leaves number, and root initiation) and fresh weight of soybean callus bioassays. Algal and cyanobacteria extracts contained hormones, vitamins, enzymes, many micro and macro nutrients which may be used as biostimulants (or biofertilizers) for different plant growth and development.
我們已成功檢測並定量由藍綠菌（米麴菌與念珠藻）及綠藻門（小球藻）物種內源性產生的植物激素——生長素與細胞分裂素（IAA 與 BA）。培養於不同濃度處理廢水結合 BG11 或 BG11 ₀ 培養基的藻類萃取物，對番茄幼苗形態參數（芽長、分枝數、葉片數及根原基形成）與大豆癒傷組織鮮重生物測定均展現顯著影響。藻類與藍綠菌萃取物所含的激素、維生素、酵素及多種微量與巨量營養素，可作為促進不同植物生長發育的生物刺激素（或生物肥料）。