CN112813004A - 一株耐紫外光抗氧化的不动细菌及其应用 - Google Patents
一株耐紫外光抗氧化的不动细菌及其应用 Download PDFInfo
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Abstract
一株耐紫外光抗氧化的不动细菌及其应用,涉及微生物领域,尤其涉及一株耐紫外光抗氧化的不动细菌及其应用。要解决现有ICPB工艺的生物膜容易受到紫外光和可见光的影响,导致死亡或受到毒害的问题。该细菌为强光催化不动杆菌(Acinetobacterenhanphotocatalysis)C3,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年11月20日,保藏编号为CGMCC No.18984。强光催化不动杆菌C3可在紫外光UV254照射条件下生长,能够强化紫外光对腐殖酸的降解效果,在紫外光UV254照射条件下经过10h处理,腐殖酸去除率可达到67.19%。本发明应用于废水处理领域。
Description
技术领域
本发明涉及微生物领域,尤其涉及一株耐紫外光抗氧化的不动细菌及其应用。
背景技术
21世纪初,Bruce E.Rittmann首先提出了光催化和生物降解直接耦合技术(Intimated Coupling of Photocatalysis and Biodegradation,ICPB)。该技术是将生物膜培养在负载有TiO2的多孔载体骨架内,防止微生物受到TiO2光催化所需的紫外线和光催化产生的羟基自由基的攻击,并且TiO2光催化降解产物的可生物降解部分会立即被生物膜降解,而不可生物降解的部分则通过光催化进一步氧化,从而实现对有机污染物的快速彻底矿化。ICPB技术提出后,引起了学者的广泛研究兴趣,主要集中在开发高效的光催化剂和光催化-生物反应器,并且大量的研究证实ICPB技术在处理有毒有机污染物方面比单纯光催化或单纯生物降解技术具有优势。但ICPB技术在使用过程中,由于紫外光照射,对载体外部生物膜产生很大的不利影响,导致载体骨架外部的微生物死亡脱落,不仅影响生物处理效果,也使水体变浑浊,削弱了紫外光的穿透力。
为了减少紫外光产生的负面效应,同时节约紫外光消耗的能源,近年来,一部分研究致力于用可见光替代紫外光。尽管可见光不会对微生物产生破坏细胞膜结构、损伤DNA等直接影响,但是由于可见光催化作用产生的羟基自由基具有极强的氧化能力,且反应迅速,可以和生物体内几乎所有的物质反应,例如糖、核酸、碱基、蛋白质等,对微生物产生毒害作用。目前,ICPB工艺的生物膜主要是利用污水厂混合污泥培养固定形成的混合菌群,微生物组成相对复杂,缺少对耐紫外光或抗氧化的专性微生物。
发明内容
本发明是要解决现有ICPB工艺的生物膜容易受到紫外光和可见光的影响,导致死亡或受到毒害的问题,提供一株耐紫外光抗氧化的不动细菌及其应用。
本发明耐紫外光抗氧化的不动细菌为强光催化不动杆菌(Acinetobacterenhanphotocatalysis)C3,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址是北京市朝阳区北辰西路1号院3号,保藏日期为2019年11月20日,保藏编号为CGMCCNo.18984。
本发明的强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3的形态特征为:球杆菌,长为0.6~1.5μm,宽为1.3~1.4μm,无鞭毛;革兰氏染色阴性,但不易脱色;无芽孢;菌落圆形,光滑,凸起。
本发明的强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3的生理生化特征:为专性好氧菌,可在15~45℃、pH 6~8条件下生长;最适生长温度为35℃,最适生长pH值为7.2,可在紫外光UV254条件下存活;接触酶阳性,氧化酶阳性,可以乙酸钠为唯一碳源进行生长,可以水解葡萄糖、吐温40,可利用丙氨酸,L-乳酸、醋酸、丙酸、α-丁酮酸、α-羟丁酸、α-酮戊二酸、苹果酸、1%乳酸钠;对洁霉素,万古霉素,利福霉素、氨曲南抗性较强。
本发明强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3的分子生物学鉴定结果:通过16S rDNA序列比对分析,与其相近种属布维不动细菌(Acinetobacterbouvetii)相似度仅为97%,G+C含量为44.29%。通过结合菌体形态特征、生长条件、生理生化鉴定结果确定,强光催化不动杆菌C3为一新种细菌。
本发明强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3在废水处理中的应用。
本发明强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3在光催化氧化中的应用。
进一步的,所述强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3在ICPB体系中的应用。
进一步的,所述强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3用于形成生物膜负载在TiO2的多孔载体骨架内。
本发明的有益效果:
本发明耐紫外光抗氧化的不动细菌,为一新种细菌,它命名为强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3,其可在紫外光UV254照射条件下生长,在35℃、紫外光条件下的世代时间为2.6min。强光催化不动杆菌C3能够强化紫外光对腐殖酸的降解效果,在紫外光UV254照射条件下经过10h处理,腐殖酸去除率可达到67.19%。
附图说明
图1为本发明强光催化不动杆菌C3的菌体形态;
图2为本发明强光催化不动杆菌C3的菌落形态;
图3强光催化不动杆菌C3在紫外光和暗培养条件下的生长曲线。
具体实施方式
本发明技术方案不局限于以下所列举具体实施方式,还包括各具体实施方式间的任意组合。
具体实施方式一:本实施方式耐紫外光抗氧化的不动细菌为强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址是北京市朝阳区北辰西路1号院3号,保藏日期为2019年11月20日,保藏编号为CGMCC No.18984。
具体实施方式二:本实施方式耐紫外光抗氧化的不动细菌C3的筛选方法:从黑龙江大学校园内花坛中取300mg表层土壤,加入蒸馏水至1L,搅拌均匀后静置。静置后的上清液为土壤浸出液。取3份300mL土壤浸出液分别加入500mL筛选液,在35℃、UV254紫外灯照射磁力搅拌器下培养3天。经过梯度稀释后,在35℃、UV254紫外灯照射条件下进行划线分离,选取典型的单一菌落进行纯化培养,即为本实施方式的菌株C3。
所述筛选液成分为:1g/L碳源,NH4Cl 0.1~0.5g/L,MgSO4·7H2O 0.01~0.05g/L,K2HPO4 0.05~0.2g/L,NaCl 0.05~0.12g/L,MnSO4·4H2O 0.01g/L,FeSO4 0.01g/L,0.5~1g/L筛选用催化剂,其中碳源为腐殖酸、葡萄糖、淀粉或乙酸钠,筛选用催化剂可以是TiO2、铁粉、硫酸亚铁等。
本实施方式参照《伯杰细菌鉴定手册》第八版和《常见细菌系统鉴定手册》对耐紫外光抗氧化的不动细菌C3进行生理生化鉴定:
菌株的形态特征为:球杆菌,如图1所示,长为0.6~1.5μm,宽为1.3~1.4μm,无鞭毛;革兰氏染色阴性,但不易脱色;无芽孢;菌落圆形,光滑,凸起,如图2所示。
生理生化特征为:专性好氧菌,可在15~45℃、pH 6~8条件下生长;最适生长温度为35℃,最适生长pH值为7.2,可在紫外光UV254条件下存活;接触酶阳性,氧化酶阳性,可以乙酸钠为唯一碳源进行生长,可以水解葡萄糖、吐温40,可利用丙氨酸,L-乳酸、醋酸、丙酸、α-丁酮酸、α-羟丁酸、α-酮戊二酸、苹果酸、1%乳酸钠;对洁霉素,万古霉素,利福霉素、氨曲南抗性较强。
对菌株C3进行分子鉴定,提取细菌基因组DNA后,进行16S rDNA序列扩增,用于扩增的PCR反应引物为通用引物:正向引物5’-GAGCGGATAACAATTTCACACAGG-3’;反向引物5’-CGCCAGGGTTTTCCCAGTCACGAC-3’。PCR反应体系如下:模板DNA 50~100ng,25μL Taq酶混合液,0.5μL正向引物,0.5μL反向引物,加入无菌去离子水至50μL。PCR扩增条件:94℃预变形5min,之后94℃变性1min,50~55℃扩增1min,72℃延伸1.5min,经过30次循环后,72℃延伸5min。将获得片段用于测序。获得1473bp序列长度,提交至GenBank注册号为MN587977,16SrDNA序列如序列表中SEQ ID NO:1所示。
与其相近种属布维不动细菌(Acinetobacter bouvetii)相似度仅为97%,G+C含量为44.29%,通过结合菌体形态特征、生长条件、生理生化鉴定结果确定,本实施方式的菌株C3为一新种细菌,命名为强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3。
具体实施方式三:本实施方式强光催化不动杆菌(Acinetobacterenhanphotocatalysis)C3在紫外光UV254照射条件下的生长试验:
紫外灯光254nm,在紫外灯光强27.8μW/cm2、35℃的条件下,用标准LB培养基(胰蛋白胨10g/L,酵母提取物5g/L,NaCl 10g/L)培养菌株Acinetobacter enhanphotocatalysisC3,通过测定溶液在600nm下的吸光度值(OD600)来绘制菌株的生长曲线,并与在无光照、35℃条件下培养的菌株C3生长曲线做对比,结果如图3所示,图中●表示紫外光条件,■表示暗培养条件。紫外灯UV254照射条件下,菌株C3生长的适应期缩短,这与紫外光分解水中有机物,使有机物更容易被菌株利用有关。在紫外条件下培养24h后,菌株C3的生长呈稳定、衰亡趋势,与暗培养条件相似,说明长时间的紫外照射对菌株伤害很小,生长呈衰亡趋势主要是由于培养基中营养不足,限制了菌体生长,由此可见,在紫外光强27.8μW/cm2条件下照射培养有利于菌株C3的生长。
本实施方式强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3可在紫外光UV254照射条件下生长,在35℃、紫外光条件下的世代时间为2.6min。
具体实施方式四:本实施方式本实施方式强光催化不动杆菌(Acinetobacterenhanphotocatalysis)C3在紫外光UV254照射条件下去除水中腐殖酸的试验:
取0.3~0.5mg的新培养C3菌体,接种至100mL腐殖酸溶液中,在35℃、紫外灯光254nm、紫外灯光强27.8μW/cm2、160rpm的条件下,对腐殖酸处理10h之后测定水中总有机碳(TOC)浓度,以未接种菌体的腐殖酸溶液为对照,并分析菌株C3对腐殖酸的去除效果,结果如表1所示。
表1强光催化不动杆菌C3在紫外光催化条件下对腐殖酸的去除效果
结果显示,未接种菌体的处理中,TOC去除效果很低,不到10%。而经过菌株C3处理10h,可以去除水中67.19%的腐殖酸。
紫外光照射可以将腐殖酸初步分解成为可生物降解的小分子有机物,而菌C3能够耐紫外线照射,并能够利用可生物降解有机物进行生长代谢,将其彻底氧化,因而使水中的有机物达到了彻底分解的作用。因此本发明的菌株C3能够强化紫外光对腐殖酸的降解效果。
序 列 表
<110> 黑龙江大学
<120>一株耐紫外光抗氧化的不动细菌及其应用
<160> 3
<210> 1
<211> 1473
<212> DNA
<213>强光催化不动杆菌(Acinetobacter enhanphotocatalysis)
<220>
<223>强光催化不动杆菌C3
<400> 1
ggctcagatt gaacgctggc ggcaggctta acacatgcaa gtcgagcgga gttgtggtgc 60
ttgcaccata acttagcggc ggacgggtga gtaatgctta ggaatctgcc tattagtggg 120
ggacaacatc tcgaaaggga tgctaatacc gcatacgtcc tacgggagaa agcaggggac 180
cttcgggcct tgcgctaata gatgagccta agtcggatta gctagttggt ggggtaaagg 240
cctaccaagg cgacgatctg tagcgggtct gagaggatga tccgccacac tgggactgag 300
acacggccca gactcctacg ggaggcagca gtggggaata ttggacaatg gggggaaccc 360
tgatccagcc atgccgcgtg tgtgaagaag gccttttggt tgtaaagcac tttaagcgag 420
gaggaggcta ctggtattaa tactaccgga tagtggacgt tactcgcaga ataagcaccg 480
gctaactctg tgccagcagc cgcggtaata cagagggtgc gagcgttaat cggatttact 540
gggcgtaaag cgtgcgtagg cggcttttta agtcggatgt gaaatccctg agcttaactt 600
aggaattgca ttcgatactg ggaggctaga gtatgggaga ggatggtaga attccaggtg 660
tagcggtgaa atgcgtagag atctggagga ataccgatgg cgaaggcagc catctggcct 720
aatactgacg ctgaggtacg aaagcatggg gagcaaacag gattagatac cctggtagtc 780
catgccgtaa acgatgtcta ctagccgttg gggcctttga ggctttagtg gcgcagctaa 840
cgcgataagt agaccgcctg gggagtacgg tcgcaagact aaaactcaaa tgaattgacg 900
ggggcccgca caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa gaaccttacc 960
tggtcttgac atacagagaa ctttccagag atggattggt gccttcggga actctgatac 1020
aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080
gcgcaaccct tttccttact tgccatcggg tcatgccggg aactttaagg atactgccag 1140
tgacaaactg gaggaaggcg gggacgacgt caagtcatca tggcccttac gaccagggct 1200
acacacgtgc tacaatggtc ggtacaaagg gttgctacct agcgatagga tgctaatctc 1260
aaaaagccga tcgtagtccg gattggagtc tgcaactcga ctccatgaag tcggaatcgc 1320
tagtaatcgc ggatcagaat gccgcggtga atacgttccc gggccttgta cacaccgccc 1380
gtcacaccat gggagtttgt tgcaccagaa gtaggtagtc taaccgcaag gaggacgctt 1440
accacggtgt ggccgatgac tggggtgaag tcg 1473
<210> 2
<211> 24
<212> DNA
<213>人工序列
<220>
<223> 正向引物
<400> 2
gagcggataacaatttcacacagg 24
<210> 3
<211> 24
<212> DNA
<213>人工序列
<220>
<223> 反向引物
<400> 3
cgccagggttttcccagtcacgac 24
Claims (5)
1.一株耐紫外光抗氧化的不动细菌,其特征在于该细菌为强光催化不动杆菌(Acinetobacter enhanphotocatalysis)C3,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址是北京市朝阳区北辰西路1号院3号,保藏日期为2019年11月20日,保藏编号为CGMCC No.18984。
2.如权利要求1所述的耐紫外光抗氧化的不动细菌在废水处理中的应用。
3.如权利要求1所述的耐紫外光抗氧化的不动细菌在光催化氧化中的应用。
4.根据权利要求3所述的应用,其特征在于所述耐紫外光抗氧化的不动细菌用于ICPB体系中。
5.根据权利要求4所述的应用,其特征在于所述耐紫外光抗氧化的不动细菌用于形成生物膜负载在TiO2的多孔载体骨架内。
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