錦集
  RMS 通過厭氧氨氧化生物抑制TN的去除性能
  RMs 可以顯著提高厭氧氨氧化菌關(guān)鍵酶的活性
  RMs是推斷發(fā)揮作用的Q/QH2在厭氧氨氧化過程
  作為主要的原因，可能會(huì)阻止ladderane RMs 和關(guān)鍵酶之間的聯(lián)系
文章信息
1.文章歷史
   2013年9月修訂
   2013年10月31日修訂編版
   2013年11月1日被接受
   2013年11月10日在網(wǎng)上發(fā)布
2.關(guān)鍵詞
   厭氧氨氧化
   氧化還原介質(zhì)
   肼脫氫酶
   亞硝酸鹽還原酶
   硝酸還原酶
3.摘要
   本研究shou先探討厭氧氨氧化生物/關(guān)鍵酶和醌的氧化還原介質(zhì)之間的活動(dòng)關(guān)系，其中蒽醌-2,6-二磺酸（AQDS），2-羥基-1,4- napthoqui-無（LAW）和蒽醌-2-羧酸（AQC）。實(shí)驗(yàn)結(jié)果表明，總脫氮性能隨三種氧化還原介質(zhì)（RMS）用量的增加而呈下降趨勢。例如，當(dāng)AQC增加到0.8毫米，TN的去除率急劇減少到17.2mg-N/gVSS/h，只能控制大約20%。這種現(xiàn)象可能是微生物中毒與細(xì)胞外的RM增加而引起的。然而，粗肼脫氫酶，亞硝酸鹽還原酶，和硝酸還原酶的活性增強(qiáng)比沒有RMS的對照實(shí)驗(yàn)約0.6-3倍。RMS被推斷在厭氧氨氧化過程中發(fā)揮輔酶/泛醌（Q/QH2）作用。此外，具體ladderane 膜結(jié)構(gòu)可以阻隔RMS和厭氧氨氧化膜內(nèi)的關(guān)鍵酶。主要原因可能是RMS對厭氧氨氧化生物和關(guān)鍵酶的反向影響。
1.簡介
   厭氧氨氧化（ANAMMOX）現(xiàn)在被確認(rèn)為是一種新穎的重要的生物脫氮工藝。它可以在厭氧條件下將NH4與NO2直接轉(zhuǎn)化成N2（Strouset等人，1999）。與傳統(tǒng)工藝相比（硝化反硝化生物），厭氧氨氧化過程提供了顯著的優(yōu)點(diǎn)，如對氧氣和有機(jī)碳，低污泥產(chǎn)量和減少CO2和NO2的排放（Opden Campet等人，2006）。近日，唐等人（2010）報(bào)告了一個(gè)高達(dá)74.3-76.7 kg-N/m3/d的脫氮率在一個(gè)實(shí)驗(yàn)室規(guī)模的厭氧氨氧化UASB反應(yīng)器，在廢水生物脫氮的厭氧氨氧化工藝的高電位。然而，如此高的脫氮率（NRR）是通過連續(xù)添加厭氧氨氧化污泥到目標(biāo)反應(yīng)器，其中生物量濃度的增加高達(dá)42-57.7VSS/L（唐等人，2010）。此外，厭氧氨氧化菌相對長的培養(yǎng)時(shí)間為也會(huì)導(dǎo)致較長的啟動(dòng)時(shí)間，通過降低厭氧氨氧化菌的豐度使厭氧氨氧化系統(tǒng)更加脆弱。因此，提高生物質(zhì)厭氧氨氧化菌活性，并進(jìn)一步縮短厭氧氨氧反應(yīng)器的啟動(dòng)是很趣味性和挑戰(zhàn)性的課題。研究人員已經(jīng)進(jìn)行了大量的努力， 通過外場能量（磁場力，低強(qiáng)度超聲）或添加幾種微量營養(yǎng)元素增加厭氧氨氧化菌的活性。例如，劉等。（2008）施加的磁場成功地提高厭氧氨氧化菌的活性，在60T的磁化率下，**大脫氮率提高了30%。類似地，段等，（2011）表明，總氮（TN）厭氧氨氧化菌的去除率提高25.5%，通過施加0.3W/cm2超聲強(qiáng)度與4分鐘的**佳照射時(shí)間，這個(gè)效果可以持續(xù)6天左右。除了外部*域的應(yīng)用，喬等（2012）表明Mno2粉末的加入也可以使厭氧氨氧化菌的脫氮率為無二氧化錳粉的2倍。
近日，氧化還原介質(zhì)（RMS）被發(fā)現(xiàn)在有機(jī)和無機(jī)污染物的厭氧生物轉(zhuǎn)化中起著重要的作用（范德齊和塞萬提斯，2009）。有一些研究集中在氧化還原介質(zhì)的反硝酸化脫氮工藝中的作用。阿蘭達(dá)泰馬瑞等人（2007）通過反硝化生物，包括蒽醌-2,6-二磺酸（AQDS），1,2-萘醌-4-磺酸酯e(NQS).2,6-disulfonate(AQDS), 2-羥基-1,4-萘醌，研究了同時(shí)轉(zhuǎn)換硫化物和硝酸鹽不同醌的氧化還原介質(zhì)的影響。他們證明，NQS必須使用硫化氫作為電子供體的**高硝酸鹽還原率（荷蘭達(dá)泰馬瑞等人，2007）。Guoet等人（2010）探討了氧化還原介質(zhì)催化脫氮工藝與蒽醌(AQ)由海藻酸鈣固定。他們還發(fā)現(xiàn)，加入500個(gè)蒽醌固定化珠會(huì)加速脫氮約兩倍。劉等（2012）證明，固定到功能聚合生物載體2-磺酸蒽醌（00.4mmol/L），可以提高脫氮率約1.5倍。直到現(xiàn)在還沒有關(guān)于RMS對厭氧氨氧化生物有影響的報(bào)告。
反硝化生物量**關(guān)鍵酶位于細(xì)胞膜或細(xì)胞膜外介質(zhì)。因此，RMS可以接觸這些酶和加快硝酸鹽或亞硝酸鹽的生物降解速率。然而，厭氧氨氧化菌的關(guān)鍵酶是位于厭氧氨氧化菌膜內(nèi)，并在其膜引起質(zhì)子動(dòng)力勢和隨后的由膜結(jié)合ATP酶合成ATP（圖1中示出）。從厭氧氨氧化菌外面進(jìn)入?yún)捬醢毖趸?，RMS必須穿過細(xì)胞壁，細(xì)胞質(zhì)膜，卵胞漿內(nèi)膜和厭氧氨氧化膜以便與關(guān)鍵酶接觸。厭氧氨氧化菌膜結(jié)構(gòu)由C18和C20脂肪酸組成，包括3個(gè)或5個(gè)線性級鏈環(huán)丁烷（sinningheet等人，2002）。它們被脂結(jié)合到甘油骨架或醚結(jié)合的烷基鏈（sinningheet等人，2005）。因此，ladderane可能會(huì)阻止RMS和厭氧氨氧化膜內(nèi)部的關(guān)鍵酶之間的接觸。
本研究的目的是討調(diào)查三種RMS對厭氧氨氧化生物活性的影響。厭氧氨氧化菌的關(guān)鍵酶（肼脫氫酶硝酸還原酶、亞硝酸還原酶）上RMS的影響也進(jìn)行了研究。還討論了在兩個(gè)厭氧氨氧化生物和關(guān)鍵酶上的效果的可能機(jī)制。所測試的RMS包括蒽醌-2,6-二磺酸（AQDS），2-羥基-1,4-萘醌（法）和蒽醌-2-羧酸（AQC）。
2.方法
2.1. 微生物與資料媒體
厭氧氨氧化污泥用于接種源自形反應(yīng)器的厭氧氨氧化實(shí)驗(yàn)規(guī)模的實(shí)驗(yàn)室。反應(yīng)器的內(nèi)徑和高度分別為8和45厘米。這種反應(yīng)器在670天的操作的總脫氮（TN）率為 8.0 kg-N/m3/d 。 KSU-1株(AB057453.1)的厭氧氨氧化菌約占種子的總生物量的70–75%。在實(shí)驗(yàn)中使用的介質(zhì)主要是由在（NH4）2SO4和亞硝酸鈉中的銨和亞硝酸鹽的形式。該微量礦物質(zhì)培養(yǎng)基的組成如范德格拉夫等人描述（1996）。#p#分頁標(biāo)題#e#
2.2 批量測試
為了確定不同的RMS濃度對特殊厭氧氨氧化活性的影響，制定了七組是RMS從0到0.8毫米不同濃度的實(shí)驗(yàn)。該實(shí)驗(yàn)是在七個(gè)120毫升含100毫升培養(yǎng)基瓶的血清瓶，每個(gè)包含厭氧氨氧化的生物質(zhì)具有不同的RMS（MLVSS濃度2000 mg/L）。生物樣品取自反應(yīng)器并在礦物質(zhì)中洗滌三次，以除去殘留的氮?dú)狻H值調(diào)整**7.5，將溫度保持在35± 1 °C左右的水域搖床。搖動(dòng)轉(zhuǎn)速設(shè)定為150轉(zhuǎn)的轉(zhuǎn)速，保持生物量和媒體之間的充分接觸。血清瓶內(nèi)容物與二氮的氣體被洗清以除去溶解的氧。初始NH4 -N和NO2 -N濃度都設(shè)定在50毫克/升。特別厭氧氨氧化活性，隨著時(shí)間流逝由在瓶里的氨和亞硝酸的濃度每單位生物量降低濃度曲線峰值改變。使用無菌注射器每小時(shí)收集一次樣品，并通過0.45lm孔徑的膜吹掃來分析NH4 -N，NO2- N，NO3- N，和RMS濃度。
2.3分析方法
Concentrations of nitrite and nitrate were determined by using on-exchange chromatography (ICS-1100, DIONEX, AR, USA) with an Ion Pac AS18 anion column after ltration with 0.22 lm poresize membranes. NH4-N, MLSS and MLVSS concentrations were measured according to the Standard Methods (APHA, 1995).
硝酸鹽和亞硝酸鹽的濃度通過使用離子交換色譜測定法（ics-1100，Dionex，AR，USA）與0.22ml 孔徑膜過濾PAC AS18陰離子柱。NH4-N，MLSS和MLVSS濃度按標(biāo)準(zhǔn)方法測定（APHA，1995）。
pH值的測量是通過使用一個(gè)數(shù)字ph計(jì)（phs-25，雷磁公司，中G），而DO使用數(shù)字DO做計(jì)進(jìn)行測量（YSI，模型55，美G）。使用分光AQDS，AQC和規(guī)律濃度均在其**大吸光度采用分光光度法測量（k = 328，336，452 nm）用分光光度計(jì)（v-560uv /VIS分光光度計(jì)，日本）根據(jù)哈藤巴赫等人。（2008）和勞等人。（2002）
2.4生物質(zhì)提取物的制備及酶活性的測定
從我們的實(shí)驗(yàn)室規(guī)模的反應(yīng)器中量取5克（濕重）厭氧氨氧化生物質(zhì)。生物樣品離心8000 rpm離心轉(zhuǎn)，在4°C進(jìn)行 20 分鐘，隨后用磷酸鈉緩沖溶液洗滌兩次（20毫米，pH 7.0）。洗滌后的沉淀在20毫升緩沖液溶解再沉淀后超聲（225 W，4°C 30分鐘，超聲波處理器CPX 750，美G）。細(xì)胞團(tuán)通過離心（22000轉(zhuǎn)）分離，在4°C進(jìn)行 30分鐘。將上清液儲(chǔ)存在4°C作為蛋白質(zhì)和酶活性測定細(xì)胞提取物。蛋白質(zhì)濃度的測量根據(jù)布拉德福德（布拉德福德法，1976），以牛血清白蛋白為標(biāo)準(zhǔn)。據(jù)島村等人描述的肼脫氫酶酶活性方法測定（2007），并且反應(yīng)用分光光度計(jì)描繪為在550 納米處增加在標(biāo)注混合物中細(xì)胞色素C的吸光度（v-560紫外可見分光光度計(jì)，雅有限公司，日本）。該混合物由100毫米磷酸鉀緩沖液（pH 7.0），50lm馬心臟細(xì)胞色素C（氧化型），酶解液適量，和25lm肼。肼脫氫酶（HDH）活性表示為細(xì)胞色素減少／毫克蛋白/分鐘。硝酸還原酶（NAR）的活性測定Meincke等人（1992）通過測定亞硝酸鹽消耗方法。氯酸鈉作為電子受體。測定含有22 mm NaClO3和11毫米50毫米亞硝酸鈉鉀磷酸鹽緩沖液，pH值7。反應(yīng)通過加入酶開始。一個(gè)單位的酶活性被denedaslmol 亞硝酸鹽還原酶（NIR）的活性作為賽義德在Hira等人描述的方法的基礎(chǔ)上（2012）以減少甲基紫（MV）作為電子供體。反應(yīng)混合物含有100 毫米磷酸鉀緩沖液（pH7.0），MV（3毫米），亞硝酸鈉（6毫米）和0.1毫升的生物質(zhì)提取物在塞緊的4毫升試管內(nèi)制備。反應(yīng)是由連二亞硫酸鈉的注射開始（12毫米）。酶活性被作為亞硝酸鹽的還原。所有的測定混合物要在35± 1°C進(jìn)行。
2.5系統(tǒng)分析
所有在本文中提出的數(shù)據(jù)是一式三份，實(shí)驗(yàn)的數(shù)據(jù)的平均值。通過使用單因素方差分析每與鄧肯的多范圍檢驗(yàn)（SPSS 19），和每次形成統(tǒng)計(jì)分析的P＜0.05值被認(rèn)為是統(tǒng)計(jì)學(xué)顯著性。

英語原文
Effects of quinoid redox mediators on the activity of anammox biomass
highlights
RMs addition depressed TN removal performance by anammox biomass.
RMs could markedly enhance the key enzymes activities of anammox bacteria.
RMs was inferred to play the role as Q/QH2 during anammox process.
Ladderane as the main reason might block the contact between RMs and key enzymes.
Article info
Article history:
Received 19 September 2013
Received in revised form 31 October 2013
Accepted 1 November 2013
Available online 10 November 2013
Keywords:
Anammox
Redox mediator
Hydrazine dehydrogenase
Nitrite reductase
Nitrate reductase
abstract
This study rst explored the relationship between the activity of anammox biomass/key enzymes and quinoid redox mediators, which were anthraquinone-2,6-
disulfonate (AQDS),2-hydroxy-1,4-napthoqui-none (LAW) and anthraquinone-2-carboxylic acid (AQC). Experimental results demonstrated that the total nitrogen removal performance showed a downward trend with all three redox mediators (RMs) dosage increasing. For instance, when the AQC addition increased to 0.8 mM, the TN removal rate sharply reduced to 17.2 mg-N/gVSS/h, only about 20% of the control. This phenomenon might be caused by microbial poisoning with the extracellular RMs additions. Nevertheless, the crude hydrazine dehydroge-nase, nitrite reductase, and nitrate reductase activities were enhanced with RMs addition, about 0.6–3folds compared to the control experiments without RMs addition. The RMs was inferred to play the role as ubiquinol/ubiquinone (Q/QH2) during the anammox process. Furthermore, the specic ladderane membrane structure could block the contacting between RMs and the key enzymes inside anammox-some. This might be the main reason for the contrary effects of RMs on anammox biomass and the key enzymes.#p#分頁標(biāo)題#e#
1. Introduction
Anaerobic ammonium oxidation (anammox) process is now
recognized as a novel and important process in biological nitrogen removal, which can directly convert NO 2 to N2 gas with NH4 under anaerobic conditions (Strous et al., 1999). Compared with the conventional biological processes (nitrication–denitrication),anammox process offers signicant advantages such as no demand for oxygen and organic carbon, low sludge production and reduced
CO2 or N2O emissions (Opden Campet al., 2006).Recently, Tang
et al. (2010) reported a very high nitrogen removal rate of 74.3–76.7 kg-N/m3/d in a lab-scale anammox UASB reactor, which demonstrated high potential of anammox process in biological nitrogen removal from wastewaters. However, such a high nitrogen removalrate (NRR) was achieved through the continuous addition of anammox seed sludge into the targeted reactor, in which the biomass
concentration increased as high as 42.0–57.7 g-VSS/L (Tanget al.,2010). Furthermore, the relative long doubling time of anammox bacteria will also cause a longer startup period and make the anammox system more vulnerable with low anammox bacteria abundance. Consequently, enhancing the bacterial activity of anammox biomass and further shortening the start-up period of anammox reactors are subjects of great interest and challenge.
Researchers have made numerous efforts to increase the activity of anammox biomass by utilizing external eld energy (mag-netic eld, low intensity ultrasound) or adding some kinds of micronutrient. For instance, Liu et al. (2008) applied magnetic eld successfully to enhance the activity of anammox bacteria whereby the maximum nitrogen removal rate increased by 30% at magnetic value of 60.0 mT in long term. Similarly, Duan et al. (2011) demon-strated that total nitrogen (TN) removal rate of anammox bacteria increased by 25.5% by applying ultrasound intensity of 0.3 W/cm2 with the optimal irradiation time of 4 min, and this effect could last
for about 6 days. Besides the application of external eld, Qiaoet al. (2012) demonstrated that the addition of MnO2 powder could also increase the nitrogen removal rate of anammox biomass about 2 times as high as that without MnO2 powder addition.
Recently, redox mediators (RMs) were found to play an important role in the anaerobic transformation of organic and inorganic contaminants (Van der Zee and Cervantes, 2009).There were a few studies focused on the role of redox mediators on nitrogen removal by denitrication process. Aranda-Tamaura et al.(2007) investi-gated the impacts of different quinoid redox mediators on the
simultaneous conversion of sulphide and nitrate by denitrifying biomass, including anthraquinone-2,6-
disulfonate (AQDS), 2-hy-droxy-1,4-naphthoquinone and 1,2-naphthoquinone-4-sulphonate(NQS).They demonstrated that NQS had the highest nitrate reduc-
tion rate using sulphide as electron donor (Aranda-
Tamaura et al.2007). Guo et al. (2010) explored the possibility of redox mediator catalyzing denitrication process with anthraquinone (AQ) immo-bilized by calcium alginate. They also found that addition of 500 anthraquinone immobilization beads would accelerate the denitri-fying rate about 2 times. Liu et al. (2012) demonstrated that anthraquinone-2-sulfonate (0.04 mmol/L) immobilized into the functional electropolymerization biocarriers could increase the
denitrication rate about 1.5 folds. Until now there was no report on the effects of RMs on anammox biomass.
Most key enzymes of denitrifying biomass are located on the cell membrane or the cell membrane periplasma. Thus, RMs could contact these enzymes and accelerate the biodegradation rate of nitrate or nitrite. However, all the key enzymes of anammox bac-teria are located inside anammoxosome, and on its membrane giving rise to a proton-motive-force and subsequent ATP synthesis by
Membrane-bound ATPases (shown in Fig.1). From the outside of anammox bacteria into anammoxosome, RMs must cross cell wall,cytoplasmic membrane, intracytoplasmic membrane and anammox some membrane in order to contact with the key enzymes.
The ladderane of the anammoxosome membrane consist of C18
and C20 fatty acids including either 3 or 5 linearly concatenated cyclobutane rings (Sinninghe et al., 2002). They are ester bound to a glycerol backbone or ether bound as alkyl chains (Sinninghe et al., 2005). Therefore, the ladderane might block the contacting between RMs and the key enzymes inside anammoxosome.
The objective of this study was to investigate the effects of three kinds of RMs on the activity of anammox biomass. The effects of RMs on the key enzymes (hydrazine dehydrogenase nitrate reductase and nitrite reductase) of anammox bacteria were also studied.The possible mechanisms of effects on both anammox biomass and the key enzymes were also discussed. The tested RMs included anthraquinone-2,6-disulfonate (AQDS), 2-hydroxy-1,4
-naphtho-quinone (LAW) and anthraquinone-2-carboxylic acid (AQC).
2. Methods
2.1. Microorganisms and feed media
The anammox sludge used for inoculation originated from a laboratory-scale anammox upow column reactor in our lab. The inner diameter and height of the column-type reactor were 8 and 45 cm, respectively. The working volume of this reactor was 2 Land continuously operated under 35 ± 1 °C. The total nitrogen(TN) removal rate of this reactor reached 8.0 kg-N/m3/d during 670 days’operation.
Anammox bacteria of KSU-1 strain(AB057453.1) accounted for about 70–75% of the total biomass in seed biomass. The media used in the experiments mainly consisted of ammonium and nitrite in the form of (NH4)2SO4 and NaNO2. The composition of the trace mineral medium was as described by vander Graaf et al. (1996).#p#分頁標(biāo)題#e#
2.2. Batch experiments
In order to ascertain the effects of different RMs concentrations on specic anammox activity, seven sets of batch experiments were conducted with the RM concentration from 0 to 0.8 mM.The tests were carried out in seven 120 ml serum vials containing 100 ml medium, each containing anammox biomass (MLVSS con-centration of 2000 mg/L) with varied RMs additions. Biomass sam-ples were taken from the reactors and washed three times with
mineral medium to remove residual nitrogen. The pH was adjusted to 7.5 and the temperature was maintained at 35 ± 1 °C in a water bath shaker. The shaking speed was set at 150 rpm to keep the full contact between biomass and media. The serum bottle contents were purged with dinitrogen gas to remove dissolved oxygen. Ini-tial NH
4 -N and NO2 -N concentrations were set at 50 mg-N/L. Spe-
cic anammox activity was estimated from the peak of the curve indicated by the decrease of ammonium and nitrite concentrations per unit biomass concentration in the vials as time lapsed. The samples were collected every hour using a sterile syringe and purged through 0.45
lm pore size membranes to analyze the NH4 -N, NO2 -N, NO3 -N and RMs concentrations.
2.3. Analytical methods
Concentrations of nitrite and nitrate were determined by using ion-exchange chromatography (ICS-1100, DIONEX, AR, USA) with an IonPac AS18 anion column after ltration with 0.22lm pore size membranes. NH4-N, MLSS and MLVSS concentrations were measured according to the Standard Methods (APHA, 1995). pH measurement was done using a digital pH meter (PHS-25, Leici Company, China), while DO was measured using a digital DO meter(YSI, Model 55, USA). The concentrations of AQDS, AQC and LAW were measured spectrophotometrically at their absorbance maxi-mum (k = 328, 336 and 452 nm, respectively) using a spectropho-
tometer(V-560UV/VIS Spectrophotometer,Jasco,Japan) according to Hartenbach et al. (2008) and Rau et al. (2002).
2.4. Preparation of biomass extracts and determination of enzyme activity 
     5 g (wet weight) anammox biomass was taken from our lab-scale reactor. The biomass samples were centrifuged at 8000 rpm at 4 擄C for 20 min followed by washing twice with sodium phos-phate buffer solution (20 mM, pH 7.0). The washed pellets were then resuspended in 20 ml of the same buffer and lysed by freezing and thawing followed by sonication (225 W, at 4 擄C for 30 min,
Ultrasonic processor CPX 750, USA). Cell mass was separated by centrifugation (22 000 rpm), at 4 擄C for 30 min. The supernatant was stored at 4 擄C and used as cell extract in the determination of protein and enzyme activity. Protein concentration was measured according to the Bradford procedure (Bradford, 1976), using BSA
as a standard. Enzyme activity of hydrazine dehydrogenase was according to the methods described by Shimamura
et al. (2007), and the reactions were depicted as an increase in the absorbance of cytochrome c at 550 nm in the standard mixture using a spectrophotometer (V-560 UV/VIS Spectrophotometer, Jas-co, Japan). The mixture consisted of 100 mM potassium phosphate buffer (pH 7.0), 50lM horse heart cytochrome c (oxidized form),an appropriate amount of enzyme solution, and 25 lM hydrazine.The hydrazine dehydrogenase (HDH) activity was expressed as
lmol of cytochrome c reduced/mg protein/min. Nitrate reductase
(Nar) activity was assayed in accordance with the methods re-corded by Meincke et al. (1992) by measuring the nitrite consump-tion. NaClO3 was used as electron acceptor. Assays contained 22 mM NaClO3 and 11 mM NaNO2 in 50 mM potassium phosphate buffer, pH 7.0. The reaction was started by the addition of the en-zyme. One unit of enzyme activity was dened as lmol of nitrite oxidized/mg protein/min. Nitrite reductase (Nir) activity was as-sayed on the basis of the methods described by Hira et al. (2012)using reduced methyl viologen (MV) as electron donors. The reac-tion mixture containing 100 mM potassium phosphate buffer (pH
7.0), MV (3 mM), sodium nitrite (6 mM) and 0.1 ml biomass extracts was anaerobically prepared in a stoppered 4.0 ml cuvette. The reac-tion was started by the injection of sodium dithionite (12 mM). Aunit of enzyme activity was dened as lmol of nitrite reduced/mg protein/min. All the assay mixtures were incubated at 35 鹵 1 擄C 2.5. Statistical analysis
All the data presented in this paper were the mean values of data from triplicate experiments. Statistical analysis was per-formed by using one-way ANOVA with the Duncan’s multiple range test (SPSS 19.0) and values of p < 0.05 were considered to be statistically signicant.