CN104560738B - 还原六价铬的草酸青霉及其筛选方法 - Google Patents
还原六价铬的草酸青霉及其筛选方法 Download PDFInfo
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- CN104560738B CN104560738B CN201510017900.1A CN201510017900A CN104560738B CN 104560738 B CN104560738 B CN 104560738B CN 201510017900 A CN201510017900 A CN 201510017900A CN 104560738 B CN104560738 B CN 104560738B
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- penicillium oxalicum
- culture medium
- hexavalent chromium
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Abstract
本发明公开了一株还原六价铬的草酸青霉及其筛选方法，属于微生物技术领域。根据菌株的形态、代谢及分子生物学特征，将其命名为草酸青霉Penicillium oxalicum SL2，并于2014年10月22日保存于中国典型培养物菌种保藏管理中心，保存号为CCTCC M 2014505。该菌株能在Cr(VI)浓度为2000mg/L的PDA培养基中生长，耐受超过100g/L的NaCl盐溶液，5天时间内能完全还原培养基中357.9mg/L的Cr(VI)，可以应用于处理环境中的Cr(VI)污染。
Description
技术领域
本发明涉及一株能还原Cr(VI)的草酸青霉SL2及其筛选方法，属于微生物技术领域。
背景技术
铬污染是当前我国面临的严重环境问题。我国是世界上最大的铬渣生产国，未处理铬渣经过多年的雨水冲淋、渗透，严重污染了堆存场地环境；钢铁、电镀、皮革等涉铬行业污水和废物未经妥善处理也严重影响相关区域的水土环境。严重的铬污染引发了云南曲靖铬污染等恶性环境事件，引起了政府和社会各界的高度关注。因此，铬污染的修复治理已刻不容缓。
自然界中铬主要以Cr(VI)和Cr(III)两种价态存在。Cr(III)有很高的有机物亲和性，是人体必需的营养元素，影响糖类和脂肪的新陈代谢；而Cr(VI)易溶于水，是国际癌症研究机构(IARQ)及美国政府工业卫生学家协会(ACGIH)确认的致癌物质，被美国EPA认定为对人体危害最大的17种化学物之一。因此，将Cr(VI)还原成Cr(III)是Cr(VI)污染修复的基本思路。
Cr(VI)污染还原修复有多种方法。目前常用的修复方法主要是物理化学方法，需要大量的化学试剂和人力，成本高，易产生二次污染，因而实际应用受到了限制。Cr(VI)污染微生物修复技术具有成本低、操作简单、可原地处理、不产生二次污染等优点，因而受到广泛的重视。自1977年首次发现Cr(VI)还原菌以来，大量Cr(VI)还原菌得到分离，如Achromobacter sp.CH-1、Leucobacter sp. CRB1、Pseudochrobactrumsaccharolyticum.LY10、Aeromonas sp. KS-14等，但国内外尚未见报道能还原Cr(VI)的草酸青霉菌株。已分离的大部分Cr(VI)还原菌适应性不强，难以耐受实际污染条件下的高盐条件，限制了其在实际Cr(VI)污染修复中的应用。因此，Cr(VI)污染修复需要获取新的、能满足实际应用要求的微生物资源，为实施微生物修复提供技术支撑。
发明内容
本发明的目的在于提供一株还原六价铬的草酸青霉及其筛选方法。该菌对Cr(VI)、盐具有很高的耐受能力，能将高毒的Cr(VI)还原成低毒的Cr(III)，为Cr(VI)污染的微生物修复提供新的资源。
一株还原六价铬的草酸青霉，能在Cr(VI)浓度为2000mg/L的PDA培养基中生长，耐受超过100g/L的NaCl盐溶液，5天时间内能完全还原培养基中357.9mg/L的Cr(VI)，可以应用于处理环境中的Cr(VI)污染，将其命名为草酸青霉（Penicillium oxalicum SL2，现保存于中国典型培养物菌种保藏管理中心，保藏号为CCTCC NO: M 2014505，保藏时间为2014年10月22日，地址为：武汉市武昌珞珈山，邮编：430072。
一种还原六价铬的草酸青霉的筛选方法，包括以下步骤：
1）收集：将含Cr(VI)的PDA平板敞开盖子置于空气中5-15天，让空气中真菌孢子在上面长出菌斑；所述含Cr(VI) PDA平板的制作方法为：20g马铃薯加100ml水煮沸过滤，滤液加水至100ml后加1g葡萄糖，1.5g琼脂，115℃灭菌20min，冷却到不烫手时加经0.22μm无菌滤头灭菌的Cr(VI)溶液，使Cr(VI)终浓度为300mg/L，倒平板；
2）驯化：将长势良好的真菌接种到含Cr(VI)液体培养基中，在30℃、250rpm振荡培养箱中培养2天，使其长成菌丝小球；所述含Cr(VI)液体培养基的制作方法为：20g马铃薯加100ml水煮沸过滤，滤液加水至100ml后加1g葡萄糖，115℃灭菌20min，冷却后加经0.22μm无菌滤头灭菌的Cr(VI)溶液，使Cr(VI)终浓度为300mg/L；
3）分离：将步骤2）中长势良好的菌丝小球接种到含Cr(VI)的PDA平板上，在30℃培养箱中倒置培养5-15天，使其长出孢子，用无菌水冲洗平板，配制浓度约为106个/mL的孢子悬浮液，按10-1、10-2、10-3、10-4、10-5稀释孢子悬浮液，各吸取0.1ml涂在含Cr(VI)浓度为300mg/L 的PDA平板上，30℃培养箱中倒置培养1-3天；
4）纯化：挑取单菌落接种到含Cr(VI)的PDA平板上，在30℃培养箱中倒置培养5-15天，使其长出孢子，用无菌水冲洗平板，配制浓度约为106个/mL的孢子悬浮液，按10-1、10-2、10-3、10-4、10-5稀释孢子液，各吸取0.1ml涂在含Cr(VI)的PDA平板上，30℃培养箱中倒置培养1-3天，得到具有高浓度Cr(VI)耐受能力的菌株。
本发明的有益效果：
本发明所提供的Cr(VI)还原菌株为草酸青霉Penicillium oxalicum SL2，该菌株能在Cr(VI)浓度为2000mg/L的PDA培养基中生长，耐受超过100g/L的NaCl盐溶液，5天时间内能完全还原培养基中357.9mg/L的Cr(VI)，可以应用于处理环境中的Cr(VI)污染。
附图说明
附图1是SL2平板菌落观察照片；
附图2是SL2显微镜（400X）观察照片；
附图3是SL2在含NaCl浓度为0、20、100g/L培养基中生长的生物量；
附图4是SL2在含Cr(VI)浓度为2000mg/L培养基中生长的生物量；
附图5是SL2对Cr(VI)的还原作用。
具体实施方式
实施例1：一株还原Cr(VI)的草酸青霉的筛选方法
1.1制作含Cr(VI)的PDA平板：称取20g马铃薯加入100ml水中，煮30min，双层纱布过滤，补水至100ml，加1g葡萄糖，1.5g琼脂，115℃灭菌20min，冷却到不烫手，加入经0.22μm无菌滤头灭菌的重铬酸钾溶液，使培养基中含300mg/L的Cr(VI)，在无菌操作台内倒平板。
1.2 制作含Cr(VI)的液体培养基：称取20g马铃薯加入100ml水中，煮30min，双层纱布过滤，补水至100ml，加1g葡萄糖，115℃灭菌20min，冷却到不烫手，加入经0.22μm无菌滤头灭菌的重铬酸钾溶液，使培养基中含300mg/L的Cr(VI)。
1.3 收集：将含Cr(VI)的PDA平板敞开盖子置于空气中5-15天，让空气中真菌孢子在上面长出菌斑。
1.4 驯化：在无菌操作台中将长势良好的真菌接种到含Cr(VI)液体培养基中，在30℃、250rpm振荡培养箱中培养2天，使其长成菌丝小球。
1.5分离：将步骤1.4驯化好的菌丝小球接种到含Cr(VI)的PDA平板上，在30℃培养箱中倒置培养10天，使其长出孢子，用无菌水冲洗平板，配制浓度约为106个/mL的孢子悬浮液，按10-1、10-2、10-3、10-4、10-5稀释孢子悬浮液，各吸取0.1ml涂在含Cr(VI) PDA平板上，30℃培养箱中倒置培养1-3天。
1.6纯化：挑取单菌落接种到含Cr(VI)的PDA平板上，在30℃培养箱中倒置培养5-15天，使其长出孢子，用无菌水冲洗平板，配制浓度约为106个/mL的孢子悬浮液，按10-1、10-2、10-3、10-4、10-5稀释孢子液，各吸取0.1ml涂在含Cr(VI)的PDA平板上，30℃培养箱中倒置培养1-3天，重复3次，得到具有高浓度Cr(VI)耐受能力的菌株，命名为SL2。
实施例2：菌株SL2的形态、碳源利用、分子生物学鉴定
菌株SL2的检测鉴定委托中国典型培养物保藏中心实施，结果如下：
2.1形态学特征
菌株SL2菌落开展，绒状。暗绿色（图1）。分生孢子链集成圆柱形，分生孢子椭圆形P（图2）。
2.2碳源利用特征
用biolog FF板检测SL2对95种碳源的利用情况，结果表明其能利用其中的56种。
表1 SL2菌株的碳源利用
| 检测项目 | 结果 | 检测项目 | 结果 | ||
| A1 | 水 | B7 | D-半乳糖 | ＋ | |
| A2 | 吐温80 | ＋ | B8 | D- 半乳糖醛酸 | ＋ |
| A3 | N-乙酰-半乳糖胺 | － | B9 | 龙胆二糖 | ＋ |
| A4 | N-乙酰-葡萄糖胺 | ＋ | B10 | D-葡萄糖酸 | ＋ |
| A5 | N-乙酰-甘露糖胺 | － | B11 | D- 葡萄糖胺 | － |
| A6 | 核糖醇 | w | B12 | α-D-葡萄糖 | ＋ |
| A7 | 苦杏仁甙 | ＋ | C1 | 1-磷酸-葡萄糖 | － |
| A8 | D- 阿拉伯糖 | － | C2 | 葡糖醛酰胺 | － |
| A9 | L- 阿拉伯糖 | ＋ | C3 | D-葡萄糖醛酸 | ＋ |
| A10 | D-阿拉伯糖醇 | ＋ | C4 | 甘油 | ＋ |
| A11 | 熊果苷 | ＋ | C5 | 糖原 | ＋ |
| A12 | D-纤维二糖 | ＋ | C6 | M-肌醇 | － |
| B1 | α- 环糊精 | － | C7 | 2-酮基-D-葡萄糖酸 | ＋ |
| B2 | β- 环糊精 | ＋ | C8 | α-D-乳糖 | ＋ |
| B3 | 葡聚糖 | ＋ | C9 | 乳果糖 | ＋ |
| B4 | I-赤丝藻醇 | ＋ | C10 | 麦芽糖醇 | ＋ |
| B5 | D-果糖 | ＋ | C11 | 麦芽糖 | ＋ |
| B6 | L-岩藻糖 | － | C12 | 麦芽三糖 | ＋ |
| D1 | D-甘露醇 | ＋ | F7 | P-邻羟基苯乙酸 | － |
| D2 | D-甘露糖 | ＋ | F8 | α-酮戊二酸 | － |
| D3 | D-松三糖 | ＋ | F9 | D-乳酸甲酯 | － |
| D4 | D-蜜二糖 | ＋ | F10 | L-乳酸 | － |
| D5 | α-甲基-D-半乳糖苷 | － | F11 | D-苹果酸 | － |
| D6 | β-甲基-D-半乳糖苷 | ＋ | F12 | L-苹果酸 | ＋ |
| D7 | α-甲基-D-葡萄糖苷 | ＋ | G1 | D-葡糖二酸 | － |
| D8 | β-甲基-D-葡萄糖苷 | ＋ | G2 | 癸二酸 | － |
| D9 | 异麦芽酮糖 | ＋ | G3 | 琥珀酰胺酸 | ＋ |
| D10 | D-阿洛酮糖 | － | G4 | 琥珀酸 | w |
| D11 | D-棉子糖 | ＋ | G5 | 琥珀酸单甲酯 | － |
| D12 | L-鼠李糖 | － | G6 | N-乙酰-L-谷氨酸 | － |
| E1 | D-核糖 | ＋ | G7 | 丙酸胺 | ＋ |
| E2 | 水杨苷 | ＋ | G8 | L-丙氨酸 | ＋ |
| E3 | 景天庚酫聚糖 | － | G9 | L-丙氨酰甘氨酸 | w |
| E4 | D-山梨醇 | ＋ | G10 | L-天冬酰胺 | ＋ |
| E5 | L- 山梨糖 | － | G11 | L-天（门）冬氨酸 | w |
| E6 | 水苏糖 | ＋ | G12 | L-谷氨基酸 | ＋ |
| E7 | 蔗糖 | ＋ | H1 | L-甘氨酰谷氨酸 | － |
| E8 | D-塔格糖 | － | H2 | L-鸟氨酸 | － |
| E9 | D-海藻糖 | ＋ | H3 | L-苯基丙氨酸 | － |
| E10 | 松二糖 | ＋ | H4 | L-脯氨酸 | ＋ |
| E11 | 木糖醇 | ＋ | H5 | L-焦谷氨酸 | － |
| E12 | D- 木糖 | ＋ | H6 | L-丝氨酸 | ＋ |
| F1 | γ-丁胺酸 | ＋ | H7 | L-苏氨酸 | － |
| F2 | 溴代丁二酸 | － | H8 | 2-乙醇胺 | － |
| F3 | 反丁烯二酸 | － | H9 | 腐胺 | － |
| F4 | β-羟基异丁酸 | － | H10 | 腺苷 | － |
| F5 | γ-羟基异丁酸 | － | H11 | 尿苷 | － |
| F6 | L-鼠李糖 | － | H12 | 腺嘌呤核苷酸 | － |
+：阳性反应； -：阴性反应；
2.3 分子生物学特征
提取SL2的总DNA，用PCR扩增目的基因rDNA-ITS（引物ITS1:5’-TCC GTA GGT GAACCT GCG G-3’；ITS4:5’-TCC TCC GCT TAT TGA TAT GC-3’）、26S rRNA基因（引物NL1:5’-GCA TAT CAA TAA GCG GAG GAA AAG- 3’；NL4:5’-GGT CCG TGT TTC AAG ACG G-3’）、18SrRNA基因（引物NS1:5’-GTA GTC ATA TGC TTG TCT C- 3’；NS8:5’-TCC GCA GGT TCA CCTACG GA- 3’）。扩增产物序列如下：
1）rDNA-ITS基因序列如SEQ ID NO:1所示。
2）26S rRNA基因序列如SEQ ID NO:２所示：
3）18SrRNA基因序列如SEQ ID NO:３所示：
将rDNA-ITS基因、26S rRNA基因、18S rRNA基因在NCBI数据库中作blast比对，结果显示该菌株与Penicilliumoxalicum某些菌株具有共同的起源，故将菌株鉴定为:草酸青霉(Penicilliumoxalicum)，命名为Penicilliumoxalicum SL2。该菌株已于2014年10月22日保存于中国典型培养物菌种保藏管理中心，保存号为CCTCC M 2014505。
实施例3：菌株SL2的耐盐、耐Cr(VI)能力检测
制作含NaCl浓度为0g/L、20g/L、100g/L的液体PDA培养基。按104个/mL接种SL2
的孢子，将培养体系放于30℃、200rpm摇床中振荡培养。每天过滤菌丝体，称重（图3）。结果表明SL2具有很强的耐盐特性，能在NaCl浓度超过100g/L的PDA培养基中生长。
在液体PDA培养基中加入经0.22μm滤膜过滤灭菌的重铬酸钾溶液，制作含Cr(VI)浓度为2000mg/L的PDA培养基。按104个/mL接种SL2的孢子，将培养体系放于30℃、200rpm摇床中振荡培养。每天过滤菌丝体，称重（图4）。结果表明SL2对Cr(VI)的耐性强，能在Cr(VI)浓度为2000mg/L的PDA培养基中生长。
实施例4：SL2对Cr(VI)的还原作用
在液体PDA培养基中加入经0.22μ.过滤灭菌的重铬酸钾和氯霉素溶液，使培养基中Cr(VI)和氯霉素溶液终浓度分别为357.9mg/L和100μg/L。按104个/mL接种SL2的孢子，将培养体系放于30℃、200rpm摇床中震荡培养。在不同的培养时间，取3ml培养液用0.22μm无菌滤头过滤，获得过滤液，用二苯碳酰二肼分光光度法检测Cr(VI) （图5）。结果显示，菌株SL2能在5天内将液体PDA培养基中的357.9mg/L Cr(VI)全部还原。
SEQUENCE LISTING
<110> 浙江大学
<120> 还原六价铬的草酸青霉及其筛选方法
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 544
<212> DNA
<213> Penicillium oxalicum SL2
<400> 1
ggtccaacct cccacccgtg tttatcgtac cttgttgctt cggcgggccc gcctcacggc 60
cgccgggggg catccgcccc cgggcccgcg cccgccgaag acacacaaac gaactcttgt 120
ctgaagattg cagtctgagt acttgactaa atcagttaaa actttcaaca acggatctct 180
tggttccggc atcgatgaag aacgcagcga aatgcgataa gtaatgtgaa ttgcagaatt 240
cagtgaatca tcgagtcttt gaacgcacat tgcgccccct ggtattccgg ggggcatgcc 300
tgtccgagcg tcattgctgc cctcaagcac ggcttgtgtg ttgggctctc gccccccgct 360
tccggggggc gggcccgaaa ggcagcggcg gcaccgcgtc cggtcctcga gcgtatgggg 420
cttcgtcacc cgctctgtag gcccggccgg cgcccgccgg cgaacaccat caatcttaac 480
caggttgacc tcggatcagg tagggatacc cgctgaactt aagcatatca ataagacgga 540
ggaa 544
<210> 2
<211> 567
<212> DNA
<213> Penicillium oxalicum SL2
<400> 2
cggcgagtga agcggcaaga gctcaaattt gaaagctggc tccttcgggg tccgcattgt 60
aatttgcaga ggatgcttcg ggagcggccc ccatctaagt gccctggaac gggccgtcat 120
agagggtgaa aatcccgtct gggatggggt gtccgcgccc gtgtgaagct ccttcgacga 180
gtcgagttgt ttgggaatgc agctctaaat gggtggtaaa tttcatctaa agctaaatat 240
tggccggaga ccgatagcgc acaagtagag tgatcgaaag atgaaaagca ctttgaaaag 300
agagttaaac agcacgtgaa attgttgaaa gggaagcgct tgcgaccaga ctcgcccacg 360
gggttcagcc ggcattcgtg ccggtgtact tccccgcggg cgggccagcg tcggtttggg 420
cggccggtca aaggccctcg gaatgtaacg ccccccgggg cgtcttatag ccgagggtgc 480
catgcggcca gcccagaccg aggaacgcgc ttcggctcgg acgctggcat aatggtcgta 540
agcgacccgt cttgaacccc ggaccaa 567
<210> 3
<211> 1670
<212> DNA
<213> Penicillium oxalicum SL2
<400> 3
gcactttata ctgtgaaact gcgaatggct cattaaatca gttatcgttt atttgatagt 60
accttactac atggatacct gtggtaattc tagagctaat acatgctaaa aaccccgact 120
tcaggaaggg gtgtatttat tagataaaaa accaacgccc ttcggggctc cttggtgaat 180
cataataact taacgaatcg catggccttg cgccggcgat ggttcattca aatttctgcc 240
ctatcaactt tcgatggtag gatagtggcc taccatggtg gcaacgggta acggggaatt 300
agggttcgat tccggagagg gagcctgaga aacggctacc acatccaagg aaggcagcag 360
gcgcgcaaat tacccaatcc cgatacgggg aggtagtgac aataaatact gatacggggc 420
tcttttgggt ctcgtaattg gaatgagaac aatttaaatc ccttaacgag gaacaattgg 480
agggcaagtc tggtgccagc agccgcggta attccagctc caatagcgta tattaaagtt 540
gttgcagtta aaaagctcgt agttgaacct tgggtctggc tggccggtcc gcctcaccgc 600
gagtactgtc cggctggacc tttccttctg gggaacctca tggccttcac tggctgtggg 660
gggaaccagg acttttactg tgaaaaaatt agagtgttca aagcaggcct ttgctcgaat 720
acattagcat ggaataatag aataggacgt gcggttctat tttgttggtt tctaggaccg 780
ccgtaatgat taatagggat agtcgggggc gtcagtattc agctgtcaga ggtgaaattc 840
ttggatttgc tgaagactaa ctactgcgaa agcattcgcc aaggatgttt tcattaatca 900
gggaacgaaa gttaggggat cgaagacgat cagataccgt cgtagtctta accataaact 960
atgccgacta gggatcggac gggattctat gatgacccgt tcggcacctt acgagaaatc 1020
aaagtttttg ggttctgggg ggagtatggt cgcaaggctg aaacttaaag aaattgacgg 1080
aagggcacca caaggcgtgg agcctgcggc ttaatttgac tcaacacggg gaaactcacc 1140
aggtccagac aaaataagga ttgacagatt gagagctctt tcttgatctt ttggatggtg 1200
gtgcatggcc gttcttagtt ggtggagtga tttgtctgct taattgcgat aacgaacgag 1260
acctcggccc ttaaatagcc cggtccgcat ttgcgggccg ctggcttctt agggggacta 1320
tcggctcaag ccgatggaag tgcgcggcaa taacaggtct gtgatgccct tagatgttct 1380
gggccgcacg cgcgctacac tgacagggcc agcgagtaca tcaccttggc cgagaggtct 1440
gggtaatctt gttaaaccct gtcgtgctgg ggatagagca ttgcaattat tgctcttcaa 1500
cgaggaatgc ctagtaggca cgagtcatca gctcgtgccg attacgtccc tgccctttgt 1560
acacaccgcc cgtcgctact accgattgaa tggctcagtg aggccttcgg actggctcag 1620
gagggttggc aacgaccccc cagagccgga aagttggtca aactcggtca 1670
Claims (1)
1.一株还原六价铬的草酸青霉（Penicillium oxalicum）SL2，其特征在于，能在Cr(VI)浓度为2000mg/L的PDA培养基中生长，耐受超过100g/L的NaCl盐溶液，5天时间内能完全还原培养基中357.9mg/L的Cr(VI)，应用于处理环境中的Cr(VI)污染，保存于中国典型培养物菌种保藏管理中心，保藏号为CCTCC NO: M 2014505。
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