CN113930360A - 一种不动杆菌及其降解吲哚的方法和应用 - Google Patents
一种不动杆菌及其降解吲哚的方法和应用 Download PDFInfo
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- CN113930360A CN113930360A CN202111218773.3A CN202111218773A CN113930360A CN 113930360 A CN113930360 A CN 113930360A CN 202111218773 A CN202111218773 A CN 202111218773A CN 113930360 A CN113930360 A CN 113930360A
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Abstract
本发明提供一种降解吲哚的不动杆菌WWZ,保藏编号:CGMCC No.22538。本发明菌株WWZ能以比现有菌株更高的速率以吲哚唯一碳源生长并降解吲哚,不但拥有较好的底物广谱性,且在苯、苯酚、萘等常见有机污染物与吲哚共存的情况下也能生长并降解吲哚,说明对于复杂的实际生境有更强的适应能力;当生长环境中含有葡萄糖等优质碳源时,对于吲哚的降解速率相较于现有技术会有更大的提升;对于生长环境中氧气的含量具有很强的耐受性,对于实际处理废水中溶解氧的变化具有抵抗力。
Description
技术领域
本发明涉及微生物技术领域,更具体地说,涉及一种不动杆菌及其降解吲哚的方法和应用。
背景技术
吲哚是一种氮杂环芳香族化合物,通常由细菌(如E.coli等)通过色氨酸酶(TnaA)转化色氨酸产生。吲哚广泛存在于动物肠胃和植物根际,由于其一般产生于稳定期,也被视为细菌的一项生理生化指标。长期以来,吲哚被认为是一种典型的氮杂环芳香族污染物,具有毒性和潜在的致突变性能,能引起动物溶血、血红蛋白尿肾病、暂时性皮肤刺激、肿瘤形成和慢性关节炎症等。此外,吲哚还可以诱发细菌的膜毒性和氧化毒性,通过调节膜电位阻止细胞***,抑制三磷酸腺苷的产生和蛋白质折叠,引起可修复的DNA损伤等。近年来的研究发现,吲哚还可以作为一种种内、种间甚至跨界的信号分子,对微生物、植物或动物的行为进行调控。对于微生物而言,吲哚可以影响其毒力、生物膜形成、抗生素耐受性、耐酸性、耐热性、持久性形成等,还可以对菌群的群体感应产生影响;对于植物而言,吲哚可以调控植物的生长和防御***;对于动物,吲哚可以调控动物的氧化应激、肠道炎症和激素分泌,对人类的代谢型疾病如二型糖尿病有着一定的影响。
随着煤炭开采、石油加工和烟草工业的发展,大量吲哚出现在排放的废水和废气之中,从而对环境造成一定的污染。此外,世界各地畜牧业的发展造成了严重的动物粪便污染和难闻的气味,其中由肠道厌氧菌产生的吲哚和粪臭素会大量挥发。牲畜废水排放和农用土地上的生物固体应用而释放到环境中,被认为是造成牲畜污染的主要原因。一方面,吲哚具有毒性和“三致”效应等,会对人类的健康安全带来直接威胁;另一方面,吲哚作为信号分子的调控作用,还会导致废水中微生物生理特性的转变,比如增强致病病原菌的毒力、抗生素耐受性、生物膜形成能力等等,间接增加了处理废水时抗生素的使用量,导致了出水水质的降低,造成环境污染。因此,在废水处理时,需要通过一定的技术手段将其中的吲哚去除。
利用微生物降解吲哚是目前一个主流的研究方向。吲哚在产吲哚细菌中是稳定的,但许多非产吲哚细菌和真核生物可以利用多种加氧酶修饰或降解吲哚(如萘双加氧酶(NDO)、多组分苯酚羟化酶(mPH)、细胞色素P450单加氧酶等),将吲哚从体系中完全去除。吲哚的降解分为好氧降解和厌氧降解,但都要经过羟基化这一必要步骤,且在这两种降解条件下,靛红、二羟基吲哚和邻氨基苯甲酸都是常见的中间体。不同之处在于,在好氧条件下,吲哚第一步通常在C3或C2/C3位进行有氧氧化;而在厌氧条件下,吲哚通常在C2位羟基化,产生二羟基吲哚。吲哚的厌氧降解通常在产甲烷、硫酸盐还原和反硝化这三种条件下进行。相比之下,好氧降解通常有着更高的速率。本课题组前期从吲哚驯化的活性污泥样品中分离出了Cupriavidus sp.SHE,菌株SHE在30℃和150r/min的无机盐培养基中可以利用吲哚作为唯一碳源生长,并在24h内降解吲哚生成靛红、靛红酸和邻氨基苯甲酸盐。此外,部分吲哚降解菌还可以将吲哚转化为靛蓝,将其资源化,用于印染、食品、制药和半导体工业。相比需要利用有毒化合物(苯胺等)作为底物并产生工业废水的化学合成,生物合成靛蓝具有环境友好的优点,因而引起研究者广泛关注。
Acinetobacter是油类物质污染环境微生物群落中的重要组成成分,最早被研究用于石油降解,也有研究表明Acinetobacter菌株能够降解多种芳香化合物,如二苯并噻吩、二苯并呋喃等。此外,Acinetobacter菌株具有合成靛蓝色素的能力,是除了Pseudomonas外另一种著名的产靛蓝菌属,如Doukyu等从土壤样品中获得了Acinetobactersp.ST-550,能在二苯甲烷-水两相体系中降解高浓度吲哚并产生292mg/L的靛蓝。本发明筛选得到的Acinetobacter sp.WWZ能以吲哚为唯一碳源进行生长,并且以较高的效率完全降解吲哚产生靛蓝,证明该菌在吲哚废水处理中具有潜在应用前景。
发明内容
本发明提供了一种不动杆菌及降解吲哚的方法,以解决废水中吲哚难以处理的问题。
为了实现上述目的,本发明提供一株不动杆菌(Acinetobacter pittii)WWZ,其特征在于:保藏编号为CGMCC No.22538。
所述不动杆菌(Acinetobacter pittii)WWZ降解吲哚的方法,包括:
在pH值为6-11、温度为19.5-40.5℃及碳源的条件下将所述不动杆菌(Acinetobacter pittii)WWZ接种并培养,以实现对吲哚的降解。
优选方式下,所述培养不动杆菌(Acinetobacter pittii)WWZ方法包括在无机盐培养基中加入浓度2g/L的(NH4)2SO4、浓度2g/L的KH2PO4、浓度3.28g/L的Na2HPO4·12H2O、浓度0.05g/L的FeCl3·6H2O及去离子水进行培养。
优选方式下,所述pH值为7-9;所述温度为29.5-30.5℃。
优选方式下,接种和培养过程中转速为145-155r/min。
优选方式下,所述碳源包括吲哚及色氨酸、柠檬酸钠、靛红、苯酚、菲、邻苯二酚、苯中的一种。
优选方式下,所述碳源还包括外加碳源,所述外加碳源包括1g/L的葡萄糖、酵母浸粉中的一种。
所述不动杆菌(Acinetobacter pittii)WWZ在以下任一项中的应用:
A1、用于降解吲哚的应用;
A2、用于降解色氨酸、柠檬酸钠、靛红、苯酚、菲、邻苯二酚、苯的应用;
所述不动杆菌(Acinetobacter pittii)WWZ用于制备降解吲哚的产品和制备降解色氨酸、柠檬酸钠、靛红、苯酚、菲、邻苯二酚、苯的产品。
本发明的有益效果:菌株WWZ能以比现有菌株更高的速率以吲哚唯一碳源生长并降解吲哚,不但拥有较好的底物广谱性,且在苯、苯酚、萘等常见有机污染物与吲哚共存的情况下也能生长并降解吲哚,说明对于复杂的实际生境有更强的适应能力;当生长环境中含有葡萄糖等优质碳源时,对于吲哚的降解速率相较于现有技术会有更大的提升;对于生长环境中氧气的含量具有很强的耐受性,对于实际处理废水中溶解氧的变化具有抵抗力。
保藏说明
本发明涉及的生物材料样品的保藏信息:参据的微生物(株)为WWZ,分类名为皮特不动杆菌(Acinetobacter pittii),于2021年5月17日由中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)保藏,保藏编号:CGMCC No.22538。CGMCC地址为北京市朝阳区北辰西路1号院3号。
附图说明
图1是本发明菌株WWZ的SEM图;
图2是本发明菌株WWZ的***发育树;
图3是本发明菌株WWZ在无机盐培养基中的生长-降解曲线图;
图4是不同pH下菌株WWZ对吲哚的降解率;
图5是不同温度下菌株WWZ对吲哚的降解率;
图6是不同转速下菌株WWZ对吲哚的降解率;
图7是外加不同芳烃化合物时菌株WWZ对吲哚的降解率;
图8是外加不同营养物质时菌株WWZ对吲哚的降解率;
图9是本发明菌株WWZ降解吲哚的中间产物a、b;
图10是本发明菌株WWZ降解吲哚的中间产物c;
图11是本发明菌株WWZ降解吲哚的机理推测图。
具体实施方式
下面结合具体实施例对本发明做进一步说明。
实验材料和试剂
1、菌株
菌株WWZ由发明人2020年从实验室反应器中分离获得,以吲哚作为唯一碳源对菌种进行培养,通过反复地平板涂布将其分离纯化,得到纯培养物。
2、培养基
无机盐培养基:(NH4)2SO4浓度2g/L、KH2PO4浓度2g/L、Na2HPO4·12H2O浓度3.28g/L、FeCl3·6H2O浓度0.05g/L、去离子水、pH为6.6;无机盐固体培养基包括无机盐培养基和2%琼脂。培养基均在121℃,0.15MPa高温高压灭菌20min,且加入终浓度为100mg/L的吲哚作为唯一碳源,吲哚用丙酮溶解,配置成100g/L吲哚母液后加入。实验中所用接菌量为5%(v/v),在30±0.5℃,150±5r/min条件下培养。
实施例1:不动杆菌菌株的筛选
本发明有关一株不动杆菌WWZ,将取自活性污泥反应器的污泥样品经过6次稀释平板涂布,30℃培养。选取一个长势良好的单菌落挑入液体无机盐培养基进行培养,30±0.5℃,150±5r/min条件下进行培养获得菌株WWZ的菌液。菌株WWZ的细胞形态为球杆状,平均长0.93μm,宽0.62μm。扫描电镜照片如图1,所述不动杆菌属于革兰氏阴性菌,菌体为球杆状。其16S rRNA基因序列的Genbank登录号为MW881781。
该菌株于2021年5月17日保存于中国微生物菌种保藏管理委员会普通微生物中心(北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,100101),其保藏号为:CGMCCNo.22538。
实施例2:菌株的16S rRNA基因分子鉴定
提取不动杆菌WWZ的基因组DNA,通过PCR扩增16S rRNA基因序列,利用BLAST程序对序列进行同源性对比,得到与其最相近的菌属为不动杆菌,二者16S rRNA基因序列相似性为100%,因此判定WWZ为不动杆菌。图2为所述菌株16S rRNA基因的***发育树。
实施例3:菌株WWZ在无机盐培养基中的生长-降解曲线
利用实施例1中的菌液,采用无机盐培养基在30±1℃,150±5r/min条件下培养,接菌量为5%(v/v)。每4h取样,于吸光度660nm下测定吸光度作为菌株生长量,用液相色谱测定吲哚剩余量(标准曲线y=65280x+131323,R2=0.99)。利用高效液相色谱(HPLC)定量检测培养基中的吲哚浓度。取2mL样品,用等体积的乙酸乙酯对其进行震荡萃取,静置30min后。用0.45μm有机膜进行过滤后上机进行分析。本实验采用Hypersil OD32(5μm,250nm×4.6mm)型号的HPLC色谱柱。流动相为甲醇/水,检测条件为:85%甲醇(v/v),15%超纯水(v/v),进样量10μL,流速0.5mL/min,洗脱时间8min,检测波长为265nm。菌株WWZ的生长及吲哚降解情况如图3所示,菌株WWZ生长24±1h进入稳定期,12-24h为对数生长期,吲哚于32±1h完全降解。
实施例4:菌株WWZ底物广谱性的分析
利用实施例1中的菌液,采用无机盐培养基,不添加吲哚,分别加入色氨酸、靛红、苯酚、菲、萘、联苯、邻苯二酚、柠檬酸钠、2,5-二羟基苯甲酸、2-吲哚酮、苯、甲苯、邻甲酚、对甲酚、间甲酚、邻二硝基苯、间二硝基苯、对氨基苯甲酸、对氯苯胺、3-甲基吲哚50mg/L,在30±0.5℃,150±5r/min条件下培养48h,接菌量为5%(v/v)。随后测定660nm下的吸光度。如表1所示,“+”表示菌株可以生长,“-”表示菌株不能生长。除吲哚外,菌株WWZ还能利用色氨酸、柠檬酸钠、靛红、苯酚、菲、邻苯二酚、2,5-二羟基苯甲酸、苯进行生长,具有一定的抗性;但不能利用甲苯、萘、3-甲基吲哚等化合物。
表1、WWZ菌株的底物广谱性
实施例5:不同pH下菌株WWZ对吲哚的降解率
利用实施例1中的菌液,使用NaOH和HCl溶液将无机盐培养基的pH分别调至5.0、6.0、7.0、8.0、9.0、10.0以及11.0,接种量为5%(v/v),30±0.5℃,150±5r/min条件下培养36h。随后用液相色谱测定吲哚剩余量(标准曲线y=65280x+131323,R2=0.99)。如图4所示,菌株WWZ在弱碱性环境有着较好的吲哚降解能力,在pH为7、8、9时均能完全降解吲哚,而当pH为6、10、11时菌株WWZ对于吲哚的利用能力大幅下降,仅能降解约10%的吲哚,在pH为5时更是几乎失去降解能力,说明过酸和过碱的环境都会对菌株WWZ的活性产生抑制,可能影响了菌株WWZ的细胞膜通透性、胞内酶促反应、胞内物质电离性等。
实施例6:不同温度下菌株WWZ对吲哚的降解率
利用实施例1中的菌液,采用无机盐培养基,接种量为5%(v/v),转速为150±5r/min,调整培养温度为20±0.5、25±0.5、30±0.5、35±0.5、40±0.5℃,培养36h。随后用液相色谱测定吲哚剩余量(标准曲线y=65280x+131323,R2=0.99)。如图5所示,菌株WWZ对于温度变化较为敏感,在30℃时能完全降解吲哚,在35℃降解能力下降,为72.45%。而在20、25、40℃时降解能力大幅下降,分别为17.97%、9.80%和1.87%,说明在过高或过低的温度下菌株WWZ降解吲哚的活性会受到抑制。
实施例7:不同转速下菌株WWZ对吲哚的降解率
利用实施例1中的菌液,采用无机盐培养基,接种量为5%(v/v),温度为30±0.5℃,调整转速为0±5、50±5、100±5、150±5、200±5、250±5r/min,培养36h。随后用液相色谱测定吲哚剩余量(标准曲线y=65280x+131323,R2=0.99)。如图6所示,转速对于菌株WWZ的降解能力影响较小,在所有转速下几乎都能完全降解,说明溶解氧对于菌株WWZ降解吲哚的影响不大。
实施例8:外加不同芳香化合物条件下菌株WWZ对吲哚的降解率
利用实施例1中的菌液,采用无机盐培养基,分别加入苯、苯酚、萘各50mg/L,接种量为5%(v/v),30±0.5℃,150±5r/min条件下培养36h。随后用液相色谱测定吲哚剩余量(标准曲线y=65280x+131323,R2=0.99)。如图7所示,苯对于菌株的吲哚降解能力只有轻微的抑制作用,最终降解率为91.90%。而苯酚和萘可能对于菌株有毒性,对菌株WWZ的吲哚降解有较强的抑制,吲哚降解率分别为71.73%和64.68%,与其他降解菌研究中苯酚和萘的促进作用不同,菌株WWZ可能没有对应的降解酶系,但有其他专一的吲哚降解酶。
实施例9:外加不同碳源条件下菌株WWZ对吲哚的降解率
利用实施例1中的菌液,采用无机盐培养基,分别加入葡萄糖、酵母浸粉、柠檬酸钠、色氨酸1g/L,接种量为5%(v/v),30±0.5℃,150±5r/min条件下培养12h。随后用液相色谱测定吲哚剩余量(标准曲线y=65280x+131323,R2=0.99)。如图8所示,葡萄糖和酵母浸粉均能促进菌株WWZ对于吲哚的降解能力,缩短菌株WWZ生长的停滞期,外加葡萄糖可以使菌株12h内降解51.48%的吲哚,外加酵母浸粉更是能够使菌株WWZ在12h内完全降解吲哚,推测是菌株WWZ能先利用这两种营养物质快速生长,随后对吲哚进行降解。而色氨酸和柠檬酸钠的投加反而会抑制菌株的吲哚降解能力,推测菌株WWZ对这两种物质的利用能力较差。
实施例10:菌株WWZ降解吲哚的产物
利用实施例1中的菌液,采用无机盐培养基,接种量为5%(v/v),30±0.5℃,150±5r/min条件下培养36h。于18h和36h分别取样,分别在8000r/min的条件下离心5min。利用乙酸乙酯对上清液进行震荡萃取处理。利用DMSO对离心后的沉淀进行萃取,震荡悬起后再次在8000r/min的条件下离心5min。用0.45μm有机膜对上层有机液体进行过滤后,采用液相色谱/飞行时间-质谱联用(LC/TOF-MS)分析方法进行测定。如图9和图10所示,检测到了在(M-H)-为146.0240、164.0342、261.0667的分子离子峰,通过与文献的比对,判断菌株WWZ降解吲哚的中间产物为靛红、靛蓝和少量靛红酸。通过查阅文献,鉴定菌株WWZ降解转化吲哚的机理如图11所示,分别为吲哚→3-羟基吲哚→2,3-二羟基吲哚→靛红→靛红酸和吲哚→3-羟基吲哚→靛蓝。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
序列表
<110> 大连理工大学
<120> 一种不动杆菌及其降解吲哚的方法和应用
<130> 2021
<141> 2021-10-20
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1472
<212> DNA
<213> 皮特不动杆菌(Acinetobacter pittii)
<400> 1
caggctcaga ttgaacgctg gcggcaggct taacacatgc aagtcgagcg gagagaggta 60
gcttgctacc gatcttagcg gcggacgggt gagtaatgct taggaatctg cctattagtg 120
ggggacaaca tttcgaaagg aatgctaata ccgcatacgt cctacgggag aaagcagggg 180
atcttcggac cttgcgctaa tagatgagcc taagtcggat tagctagttg gtggggtaaa 240
ggcctaccaa ggcgacgatc tgtagcgggt ctgagaggat gatccgccac actgggactg 300
agacacggcc cagactccta cgggaggcag cagtggggaa tattggacaa tgggcgcaag 360
cctgatccag ccatgccgcg tgtgtgaaga aggccttatg gttgtaaagc actttaagcg 420
aggaggaggc tactttagtt aatacctaga gatagtggac gttactcgca gaataagcac 480
cggctaactc tgtgccagca gccgcggtaa tacagagggt gcaagcgtta atcggattta 540
ctgggcgtaa agcgcgcgta ggcggctaat taagtcaaat gtgaaatccc cgagcttaac 600
ttgggaattg cattcgatac tggttagcta gagtgtggga gaggatggta gaattccagg 660
tgtagcggtg aaatgcgtag agatctggag gaataccgat ggcgaaggca gccatctggc 720
ctaacactga cgctgaggtg cgaaagcatg gggagcaaac aggattagat accctggtag 780
tccatgccgt aaacgatgtc tactagccgt tggggccttt gaggctttag tggcgcagct 840
aacgcgataa gtagaccgcc tggggagtac ggtcgcaaga ctaaaactca aatgaattga 900
cgggggcccg cacaagcggt ggagcatgtg gtttaattcg atgcaacgcg aagaacctta 960
cctggccttg acatagtaag aactttccag agatggattg gtgccttcgg gaacttatat 1020
acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080
gagcgcaacc cttttcctta tttgccagcg agtaatgtcg ggaactttaa ggatactgcc 1140
agtgacaaac tggaggaagg cggggacgac gtcaagtcat catggccctt acggccaggg 1200
ctacacacgt gctacaatgg tcggtacaaa gggttgctac ctagcgatag gatgctaatc 1260
tcaaaaagcc gatcgtagtc cggattggag tctgcaactc gactccatga agtcggaatc 1320
gctagtaatc gcggatcaga atgccgcggt gaatacgttc ccgggccttg tacacaccgc 1380
ccgtcacacc atgggagttt gttgcaccag aagtagctag cctaactgca aagagggcgg 1440
ttaccacggt gtggccgatg actggggtga ag 1472
Claims (9)
1.一株不动杆菌(Acinetobacter pittii)WWZ,其特征在于:保藏编号为CGMCCNo.22538。
2.一种权利要求1所述不动杆菌(Acinetobacter pittii)WWZ降解吲哚的方法,包括:
在pH值为6-11、温度为19.5-40.5℃及碳源的条件下将所述不动杆菌(Acinetobacterpittii)WWZ接种并培养。
3.根据权利要求2所述降解吲哚的方法,其特征在于:所述培养不动杆菌(Acinetobacter pittii)WWZ方法包括在无机盐培养基中加入浓度2g/L的(NH4)2SO4、浓度2g/L的KH2PO4、浓度3.28g/L的Na2HPO4·12H2O、浓度0.05g/L的FeCl3·6H2O及去离子水进行培养。
4.根据权利要求2所述降解吲哚的方法,其特征在于:所述pH值为7-9;所述温度为29.5-30.5℃。
5.根据权利要求2所述降解吲哚的方法,其特征在于:所述碳源包括吲哚及色氨酸、柠檬酸钠、靛红、苯酚、菲、邻苯二酚、2,5-二羟基苯甲酸、苯中的一种。
6.根据权利要求2所述降解吲哚的方法,其特征在于:所述碳源还包括外加碳源,所述外加碳源包括1g/L的葡萄糖、酵母浸粉中的一种。
7.根据权利要求2所述降解吲哚的方法,其特征在于:接种和培养过程中转速为145-155r/min。
8.权利要求1所述不动杆菌(Acinetobacter pittii)WWZ在以下任一项中的应用:
A1、用于降解吲哚的应用;
A2、用于降解色氨酸、柠檬酸钠、靛红、苯酚、菲、邻苯二酚、苯的应用。
9.根据权利要求8所述应用,其特征在于:所述不动杆菌(Acinetobacter pittii)WWZ用于制备降解吲哚的产品和制备降解色氨酸、柠檬酸钠、靛红、苯酚、菲、邻苯二酚、苯的产品。
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