王秀杰,王維奇,李 軍,王思宇,張 晶,魏 佳,趙白航

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氨氮對(duì)異養(yǎng)硝化菌sp.活性影響及動(dòng)力學(xué)特性分析

王秀杰,王維奇,李 軍*,王思宇,張 晶,魏 佳,趙白航

(北京工業(yè)大學(xué)建筑工程學(xué)院,北京 100124)

異養(yǎng)硝化菌株sp.JQ1004能夠在初始氨氮濃度為0~2000mg/L范圍內(nèi)進(jìn)行生長(zhǎng)和氮源代謝,菌株在初始氨氮濃度為2500mg/L條件下被完全抑制,無(wú)法生長(zhǎng).當(dāng)菌株在溫度為30℃, pH7.5,轉(zhuǎn)速為160r/min,初始氨氮濃度分別為100,300,500, 700,1000,1500,2000,2500mg/L條件下培養(yǎng)時(shí),菌株的最大比生長(zhǎng)速率分別為0.251, 0.308, 0.286, 0.243, 0.197, 0.115, 0.088h-1,相應(yīng)的最大比氨氮降解速率分別為1.335, 1.906, 1.859, 1.759, 1.562, 1.286, 0.965g/(gDCW·d).在高濃度氨氮和游離氨的抑制作用下,菌株的比生長(zhǎng)速率及對(duì)氨氮的比降解速率隨初始氨氮濃度的增加呈先增加后降低的趨勢(shì).3種基質(zhì)抑制動(dòng)力學(xué)模型(Haldane, Yano, Aiba模型)均能夠很好地模擬菌株隨初始氨氮濃度的生長(zhǎng)變化規(guī)律,對(duì)應(yīng)地相關(guān)系數(shù)分別為0.9944,0.9983和0.9929.由Haldane模型可知,菌株在不同初始氨氮濃度(游離氨)條件下的最大氨氮比降解速率max為2.604h-1,基質(zhì)親和系數(shù)s為22.57mg/L,基質(zhì)抑制系數(shù)i為1445.31mg/L.其中由i值遠(yuǎn)大于自養(yǎng)菌(硝化細(xì)菌及厭氧氨氧化菌等)的值,這表明異養(yǎng)硝化菌株sp.JQ1004比自養(yǎng)菌具有更強(qiáng)的抗抑制能力.另外,菌株在游離氨濃度為5.436mg/L時(shí),比生長(zhǎng)速率達(dá)到最大值0.583h-1.以上研究結(jié)果表明,菌株JQ1004在處理高氨氮廢水中具有潛在的應(yīng)用前景.

異養(yǎng)硝化；動(dòng)力學(xué)；高氨氮；氨氮抑制

近年來(lái),對(duì)高氨氮廢水的處理已成為污水處理的一個(gè)難點(diǎn).高氨氮廢水若處理不當(dāng)直接排入受納水體,會(huì)造成水體富營(yíng)養(yǎng)化等問(wèn)題,對(duì)環(huán)境造成嚴(yán)重危害.通常,將含氨氮濃度超過(guò)500mg-N/L的廢水稱為高氨氮廢水[1].高氨氮廢水主要來(lái)源于垃圾滲濾液[2]、養(yǎng)殖廢水[3]、焦化廢水[4]等.由于生物處理工藝具有能耗低、效率高等優(yōu)點(diǎn),已經(jīng)被越來(lái)越多應(yīng)用于高氨氮廢水的處理中.常用的工藝有短程硝化[5]、同步硝化反硝化(SND)[6]等以及它們的一些組合工藝.這些傳統(tǒng)生物處理工藝大多數(shù)依賴于自養(yǎng)型好氧/厭氧菌(硝化菌、厭氧氨氧化菌等),由于自養(yǎng)菌的世代周期長(zhǎng),且抗氨氮及有機(jī)碳等沖擊負(fù)荷能力差,導(dǎo)致了生物處理系統(tǒng)的不穩(wěn)定性[7].

Verstraete等[8]在1972年從自然界中成功分離出第一株異養(yǎng)硝化菌sp.之后, Robertson和Kuenen[9]又從活性污泥中分離出了一株同時(shí)兼有異養(yǎng)硝化和好氧反硝化功能的菌株(現(xiàn)已更名).在這之后越來(lái)越多異養(yǎng)硝化好氧反硝化菌株被分離出來(lái),如CF-S27[10],Y-11[11],YB[12]等.很多異養(yǎng)硝化好氧反硝化菌株具有能夠在高濃度氨氮條件下生長(zhǎng)代謝的特點(diǎn)[13].研究者們利用這一特點(diǎn)將該類異養(yǎng)硝化好氧反硝化菌應(yīng)用于高氨氮廢水的處理中.Joo等[14]將異養(yǎng)硝化菌株strain No. 4應(yīng)用于實(shí)際的養(yǎng)豬廢水處理中,并取得了很好的去除效果.Chen等[15]建立一個(gè)處理高鹽高氨氮廢水的生物脫氮系統(tǒng),并實(shí)現(xiàn)了穩(wěn)定運(yùn)行,利用PCR-DGGE技術(shù)檢測(cè)到系統(tǒng)中優(yōu)勢(shì)菌株為異養(yǎng)硝化好氧反硝化菌和.菌株等[16]被以生物添加的方式應(yīng)用于SBR工藝中處理高氨氮廢水并同樣取得了很好的效果.以上工藝的建立均表明,異養(yǎng)硝化好氧反硝化菌株在處理高氨氮廢水中具有很好的應(yīng)用前景.本研究通過(guò)批式實(shí)驗(yàn)研究了菌株在不同初始氨氮濃度條件下的生長(zhǎng)及代謝規(guī)律,并確定了氨氮及游離氨濃度對(duì)菌株的抑制動(dòng)力學(xué)模型,以期為菌株在處理高氨氮廢水中的應(yīng)用提供基礎(chǔ).

1 材料與方法

1.1 實(shí)驗(yàn)菌株和培養(yǎng)基

1.1.1 菌株來(lái)源 實(shí)驗(yàn)菌株sp. JQ1004為一株異養(yǎng)硝化好氧反硝化菌[17],分離自北京市高碑店污水廠中試設(shè)備的活性污泥,已被證實(shí)能夠在好氧條件下,以琥珀酸鈉為碳源,分別以氨氮和硝氮為氮源進(jìn)行生長(zhǎng)代謝.當(dāng)氨氮作為唯一氮源時(shí),菌株能夠迅速利用氨氮和有機(jī)碳進(jìn)行生長(zhǎng)代謝, 當(dāng)培養(yǎng)基中不添加有機(jī)碳源時(shí),菌株JQ1004無(wú)法生長(zhǎng),這表明菌株具有異養(yǎng)硝化作用.另外,菌株JQ1004能夠在好氧條件下,以硝氮為氮源進(jìn)行生長(zhǎng)代謝,這表明菌株具有好氧反硝化作用.

1.1.2 培養(yǎng)基 實(shí)驗(yàn)用培養(yǎng)基為異養(yǎng)硝化培養(yǎng)基[12],其配方為(NH4)2SO40.47g,(CH2COONa)2·6H2O4.219g, 50mL維氏鹽溶液, 950mL超純水,通過(guò)改變加入(NH4)2SO4的質(zhì)量改變培養(yǎng)基初始氨氮濃度,固定TOC/TN=7.5.維氏鹽溶液配方為K2HPO4·3H2O 6.55g, MgSO4·7H2O 2.5g, NaCl 2.5g, MnSO4·H2O 0.038g, FeSO4·7H2O 0.05g, 1000mL超純水.培養(yǎng)基在使用前用Na2HPO4和NaH2PO4緩沖溶液調(diào)整pH=7.5,121℃高壓蒸汽滅菌15min.平板培養(yǎng)基需加入1.5%~2%瓊脂粉.

1.2 批次實(shí)驗(yàn)

1.2.1 不同初始氨氮條件下菌株降解實(shí)驗(yàn) 將新鮮平板培養(yǎng)基上生長(zhǎng)茁壯的單菌落接種于液體異養(yǎng)硝化培養(yǎng)基中進(jìn)行活化,然后8000r/min離心5min收集菌體,用磷酸緩沖溶液反復(fù)洗滌3次,重懸于無(wú)菌水中作為接種液,固定接種液OD600值約0.5.菌株在不同初始氨氮濃度條件下的降解實(shí)驗(yàn)設(shè)置8個(gè)批次,初始氨氮濃度分別設(shè)定為:100,300,500,700,1000,1500,2000,2500mg/L.每個(gè)批次均設(shè)置不加接種液的空白組作為對(duì)照.培養(yǎng)條件為30℃,160r/min.定時(shí)取樣測(cè)定氨氮、硝氮、亞氮、COD濃度以及菌懸液OD600值,數(shù)據(jù)測(cè)定3次取平均值.

1.2.2 細(xì)胞濃度的測(cè)定[18]首先通過(guò)實(shí)驗(yàn)確定菌株細(xì)胞濃度(細(xì)胞干重,mg/L)與菌懸液吸光度(OD600)之間的標(biāo)準(zhǔn)曲線.以液體培養(yǎng)基做空白,在600nm波長(zhǎng)下測(cè)定菌懸液的吸光度,再根據(jù)標(biāo)準(zhǔn)曲線將OD600值換算為細(xì)胞干重(mg/L).細(xì)胞干重采用干燥法測(cè)定.

1.2.3 菌株比生長(zhǎng)速率、氨氮比降解速率、產(chǎn)率系數(shù)以及游離氨濃度的計(jì)算[19-20]

式中:為菌株的比生長(zhǎng)速率,h-1;為時(shí)刻時(shí)菌株的細(xì)胞濃度,mg/L;0為0時(shí)刻細(xì)胞濃度,mg/L;0為菌株培養(yǎng)任一階段的初始時(shí)刻,h;為菌株培養(yǎng)任一階段的結(jié)束時(shí)刻,h.

= -[(0-)/(0-)]/0(2)

式中:為比降解速率, g/(g DCW·d);0為初始時(shí)刻0氨氮濃度,mg/L;為時(shí)刻氨氮濃度, mg/L;0為0時(shí)刻菌株的細(xì)胞濃度,mg/L; DCW為細(xì)胞干重.

式中:為產(chǎn)率系數(shù), mg DCW/mg N;0為初始時(shí)刻細(xì)胞濃度, mg/L;為結(jié)束時(shí)刻細(xì)胞濃度, mg/L;0為初始時(shí)刻氨氮濃度, mg/L;為結(jié)束時(shí)刻氨氮濃度, mg/L.

1.3 分析項(xiàng)目和方法

NH4+-N:納氏試劑分光光度法; NO2--N: N-(1-萘基)-乙二胺分光光度法; NO3--N:麝香草酚分光光度法; CODcr:重鉻酸鉀法; OD600值:分光光度法; pH值、溫度: WTW/Multi3420便攜式測(cè)定儀.

2 結(jié)果與討論

2.1 不同初始氨氮濃度對(duì)菌株活性及基質(zhì)降解速率的影響

(A)~(G)初始氨氮濃度分別為100,300,500,700,1000,1500,2000mg/L

表1 不同初始氨氮濃度下菌株的比生長(zhǎng)速率及氨氮比降解速率

注:溫度為30℃, pH值約為7.5.

圖1展示了菌株在不同初始氨氮濃度條件下,菌株對(duì)碳源和氮素的降解以及菌株自身細(xì)胞濃度的變化情況.在不同初始氨氮濃度條件下菌株細(xì)胞濃度的增長(zhǎng)規(guī)律均為先經(jīng)過(guò)一定時(shí)間的遲滯期,且初始氨氮濃度越大遲滯期越長(zhǎng),之后進(jìn)入對(duì)數(shù)生長(zhǎng)期,在此時(shí)期菌株的比生長(zhǎng)速率達(dá)到最大.表1顯示了不同初始氨氮濃度條件下,菌株的最大比生長(zhǎng)速率、氨氮比降解速率以及在最大比生長(zhǎng)速率發(fā)生時(shí)間段內(nèi)菌株的產(chǎn)率系數(shù)值.由此可知,當(dāng)初始氨氮濃度設(shè)置為100~2500mg/L時(shí),菌株的比生長(zhǎng)速率隨氨氮濃度的增加而降低,并且在濃度為2500mg/L時(shí),菌株被完全抑制.相應(yīng)地,菌株對(duì)氨氮的比降解速率規(guī)律也是隨初始氨氮濃度的增加呈降低趨勢(shì).菌株在初始氨氮濃度為100mg/L時(shí),比生長(zhǎng)速率及比降解速率達(dá)到最大,分別為0.308h-1,2.021g/(g DCW·d).Zhang等[21]研究了菌株WSW-1001在初始氨氮濃度分別為5~1000mg/L時(shí),菌株的代謝規(guī)律,得到了同樣的規(guī)律,菌株在初始氨氮濃度越低時(shí),生長(zhǎng)及降解速率越快.與已報(bào)道過(guò)的異養(yǎng)硝化好氧反硝化相比,菌株JQ1004的最大比生長(zhǎng)速率大于菌株sp. S1(最大比生長(zhǎng)速率為0.186h-1)[22],菌株No.4(最大比生長(zhǎng)速率為0.2h-1)[23]等.

圖1還展示了菌株在不同初始氨氮濃度下,菌株對(duì)氨氮的降解特性.由表1可知,當(dāng)初始氨氮濃度設(shè)置為100~2500mg/L時(shí),菌株對(duì)氨氮的比降解速率隨初始氨氮濃度增加而降低,這與比生長(zhǎng)速率的變化趨勢(shì)一致.由表1可知,當(dāng)初始氨氮濃度為100,300,500,700,1000,1500,2000mg/L時(shí),菌株對(duì)氨氮的比降解速率分別為2.021,1.960, 1.859,1.759,1.562,1.286,0.965[g/g DCW·d]].另外,實(shí)驗(yàn)表明菌株可在0~2000mg NH4+-N/L條件下生長(zhǎng),在氨氮濃度為2500mg/L時(shí),菌株的生長(zhǎng)被完全抑制.許多研究已表明,異養(yǎng)硝化好氧反硝化菌能夠在高負(fù)荷氨氮濃度下進(jìn)行生長(zhǎng)繁殖.例如,Joo等[14]研究表明,菌株可在1200mg N/L的條件下生長(zhǎng);Ren等[24]研究發(fā)現(xiàn),菌株YB在1000mg N/L條件下仍然可以有較高地生長(zhǎng)速率;菌株sp. Y1-5[25]等被證實(shí)了可以在初始氨氮為1600mg N/L條件下生長(zhǎng).同時(shí),由圖1可知,菌株在進(jìn)行代謝的過(guò)程中需要較多的碳源,即培養(yǎng)基中有較高COD濃度.由圖1可知,COD濃度的變化與氨氮濃度的變化一致,即先經(jīng)過(guò)一定的緩慢遲滯期,菌株進(jìn)入對(duì)數(shù)生長(zhǎng)期后,COD濃度迅速被降解,最后進(jìn)入穩(wěn)定期后氮素和碳源降解速率緩慢降低,直到菌株停止生長(zhǎng).在菌株生長(zhǎng)過(guò)程中,均未檢測(cè)到亞氮和硝氮的濃度變化.分析原因可能為以氨氮為氮源時(shí),亞氮和硝氮作為中間產(chǎn)物在代謝過(guò)程中被迅速轉(zhuǎn)化并未產(chǎn)生積累;另一原因可能為菌株將氨氮直接轉(zhuǎn)化為氣態(tài)氮,并未轉(zhuǎn)化為硝氮或亞氮等中間產(chǎn)物.

2.2 不同初始氨氮濃度條件下菌株的降解動(dòng)力學(xué)特性分析

表2 各種動(dòng)力學(xué)模型參數(shù)的估計(jì)值

注:max為不同初始氨氮濃度條件下菌株的最大比生長(zhǎng)率;s為基質(zhì)親和常數(shù);i為基質(zhì)抑制常數(shù);為Yano模型中的常數(shù).

高負(fù)荷氨氮濃度會(huì)對(duì)菌株本身產(chǎn)生毒害作用從而抑制其對(duì)氨氮地降解.為探究最佳擬合模型,用3種不同的基質(zhì)抑制動(dòng)力學(xué)模型(Haldane模型, Aiba模型, Yano模型)來(lái)描述菌株對(duì)氨氮的比降解速率隨初始氨氮濃度(0~ 2000mg N/L)的變化,擬合后得到的動(dòng)力學(xué)參數(shù)及相關(guān)系數(shù)(R)如表2所示.由圖2可知,3種模型均能很好地模擬實(shí)驗(yàn)數(shù)據(jù),相關(guān)系數(shù)(2)分別為0.9944,0.9983和0.9929. 3種模型的變化趨勢(shì)均為菌株對(duì)氨氮的比降解速率隨氨氮濃度的上升呈先上升后下降的趨勢(shì),由Haldane模型可知,菌株JQ1004對(duì)氨氮的最大比降解速率為2.604g/(gDCW·d),發(fā)生最大比降解速率時(shí)的底物濃度可通過(guò)公式計(jì)算:

在Haldane模型中,Ks為基質(zhì)親和常數(shù),Ks值越小,則酶與底物的親和力越大,本實(shí)驗(yàn)中Ks值為22.57mg/L,這表明菌株中含有的氨氧化酶與氨氮有較高的親和力;另外,Ki為基質(zhì)抑制常數(shù), Ki值越小表明基質(zhì)對(duì)菌株的抑制作用越強(qiáng),反之,越大表示菌株的抗抑制能力越強(qiáng)[26].本研究中,異養(yǎng)硝化菌株Acinetobactor sp.JQ1004的Ki為1445.31mg/L,其值遠(yuǎn)大于自養(yǎng)硝化菌[27][Ki為(59±5)mg/L],厭氧氨氧化菌[28](Ki為67.234mg/L),這表明菌株JQ1004具有更強(qiáng)的抗抑制能力,原因可能是菌株為異養(yǎng)好氧菌,與自養(yǎng)菌相比有更短的世代周期,且對(duì)環(huán)境的耐受性更強(qiáng).

2.3 不同濃度游離氨對(duì)菌株活性的抑制作用

由Haldane模型擬合結(jié)果可知,菌株在不同初始氨氮濃度條件下對(duì)氨氮的降解呈先增加后降低的趨勢(shì).原因是在一定濃度范圍內(nèi),底物濃度的增加促進(jìn)了菌株的生長(zhǎng)代謝,當(dāng)?shù)孜餄舛冗^(guò)高時(shí)會(huì)對(duì)菌株活性產(chǎn)生抑制.而高濃度氨氮對(duì)菌株產(chǎn)生抑制的原因是氨氮在環(huán)境中形成較高濃度的游離氨,過(guò)高的游離氨濃度會(huì)對(duì)菌株活性及生長(zhǎng)產(chǎn)生抑制.為分析游離氨對(duì)菌株生長(zhǎng)的抑制規(guī)律,采用Haldane模型模擬菌株在不同濃度游離氨條件下菌株的比生長(zhǎng)速率變化情況.由圖3可知,在不同初始游離氨濃度條件下,菌株的比生長(zhǎng)速率隨初始游離氨濃度的增加呈先增加而后降低的趨勢(shì).這是由于氨氮在微堿性環(huán)境中形成了一定濃度的游離氨,較低濃度的游離氨有利于氨單加氧化酶的氧化[29],而過(guò)高濃度的游離氨會(huì)對(duì)菌株產(chǎn)生一定的抑制作用[20].基質(zhì)抑制動(dòng)力學(xué)模型Haldane模型很好地模擬了其生長(zhǎng)規(guī)律,相關(guān)系數(shù)2為0.956.由公式計(jì)算可知,菌株在游離氨濃度為

時(shí),菌株達(dá)到最大比生長(zhǎng)速率為0.583h-1.當(dāng)游離氨濃度超過(guò)該濃度時(shí)會(huì)對(duì)菌株產(chǎn)生抑制作用.

3 結(jié)論

3.1 菌株JQ1004能夠在高氨氮負(fù)荷(100~ 2000mg NH4+-N/L)條件下進(jìn)行生長(zhǎng)繁殖,并對(duì)氨氮具有一定的降解作用,在初始氨氮濃度為2500mg/L時(shí),菌株生長(zhǎng)被完全抑制.并且,菌株的生長(zhǎng)與對(duì)氨氮的代謝速率隨初始氨氮濃度增加而降低.

3.2 Haldane模型、Aiba模型以及Yano模型能夠很好地?cái)M合菌株在不同初始氨氮濃度條件下的降解動(dòng)力學(xué)特性,相關(guān)系數(shù)(2)均在0.99以上.由Haldane模型可知,菌株在初始氨氮濃度為180.61mg/L時(shí),氨氮比降解速率最大值2.604g/ (gDCW·d).由i值可知,與自養(yǎng)菌相比,異養(yǎng)硝化菌JQ1004對(duì)氨氮具有更強(qiáng)抗抑制能力.

3.3 Haldane模型同樣能夠較好的擬合初始游離氨(FA)濃度對(duì)菌株比生長(zhǎng)速率的影響,相關(guān)系數(shù)2為0.956.菌株的生長(zhǎng)隨FA濃度的增加而減慢,在FA濃度為5.436mg/L時(shí),比生長(zhǎng)速率達(dá)到最大值0.583h-1.

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Inhibition of initial ammonia and free ammonia nitrogen onsp. and their biokinetics.

WANG Xiu-jie, WANG Wei-qi, LI Jun*, WANG Si-yu, ZHANG Jing, WEI Jia, ZHAO Bai-hang

(The College of Architecture and Civil Engineering, Beijing University of Technology, Beijing 100124, China)., 2018,38(3)：943~949

The strain JQ1004 with capability of heterotrophic nitrification were able to grow and metabolism under different initial ammonium concentrations in range of 0~2000mg/L. However, the strain were completely inhibited in concentration of 2500mg/L. When strain JQ1004 was cultivated at 30℃, pH 7.5, 160rpm with the initial ammonium of 100, 300, 500, 700, 1000, 1500, 2000mg/L, the maximum specific growth rates were 0.251, 0.308, 0.286, 0.243, 0.197, 0.115, 0.088h-1, and the corresponding ammonium specific removal rates reached 1.335, 1.906, 1.859, 1.759, 1.562, 1.286, 0.965g/g (DCW·d), respectively. Due to the inhibition of free ammonia and high-strength concentration of ammonium, the specific growth rate and degradation rate of ammonia increased at first and decreased with the increase of initial concentration of ammonia nitrogen (free ammonia). Three kinetic models (Haldane, Yano, Aiba) were fitted well to the experimental growth kinetic data with the correlation coefficients (2) of 0.9944,0.9983 and 0.9929. For Haldane model, the values ofmax,s, andiwere 2.604h-1, 22.57mg/L, and 1445.31mg/L, respectively. The large values ofi, far greater than that of autotrophic nitrifiers or anaerobic ammonium-oxidizing bacteria, indicated that JQ1004 had good tolerance against high ammonium concentrations. Besides, the specific growth rate reached a maximum value of 0.583h-1when the concentration of free ammonia was 5.436mg/L.These results indicated possible future applications of JQ1004 in removing nitrogen and organic carbon from high-strength ammonium wastewater.

heterotrophic nitrification；biokinetics；high-strength ammonium；ammonia inhibition

X703.5

A

1000-6923(2018)03-0943-07

王秀杰(1991-),女,山東濰坊人,博士研究生,主要從事生物脫氮及環(huán)境分子生物學(xué)研究.發(fā)表論文2篇.

2017-08-06

國(guó)家水體污染控制與治理科技重大專項(xiàng)(2014ZX07201-011);16人才培養(yǎng)質(zhì)量建設(shè)-雙培養(yǎng)計(jì)劃新興專業(yè)建設(shè)(004000542216031)

* 責(zé)任作者, 教授, 18810925108@163.com