CN113684147A - 一种粪产碱杆菌及其应用 - Google Patents
一种粪产碱杆菌及其应用 Download PDFInfo
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- CN113684147A CN113684147A CN202110937439.7A CN202110937439A CN113684147A CN 113684147 A CN113684147 A CN 113684147A CN 202110937439 A CN202110937439 A CN 202110937439A CN 113684147 A CN113684147 A CN 113684147A
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- alcaligenes faecalis
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- 241000588813 Alcaligenes faecalis Species 0.000 title claims abstract description 41
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- B01D53/34—Chemical or biological purification of waste gases
- B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
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Abstract
本发明公开了一种Alcaligenes faecalis LYC菌株,根据其生理生化特性及16S rDNA序列将其鉴定为粪产碱杆菌属,保藏编号为:CGMCC No.21558。所述的Alcaligenes faecalis LYC负载于生物炭得到一种炭基固定化菌剂,其中的活菌含量为108cfu/g以上。所述的炭基固定化菌剂将Alcaligenes faecalis LYC固定化在生物炭载体上,提高微生物的数量及环境耐受性,应用于堆肥、垃圾、污泥或污水等污染物处理或废弃物的臭气治理中,能提高恶臭气体的脱除效率,实现生物质废弃物的资源化利用。
Description
技术领域
本发明属于生物环境技术领域,具体涉及一种粪产碱杆菌及其应用。
背景技术
随着我国城市化进程的不断扩展,生产生活中产生的废弃物所带来的恶臭问题已经成为困扰居民日常生活以及城市化发展的重要因素。恶臭污染来源广泛,成分复杂,难以定性定量分析,其发臭过程与组成结构有很大关联,需制定针对性的治理方案。因此,如何有效解决生产生活产生的废弃物所带来的恶臭问题已成为当前亟待解决的重大课题。
纵观国内外在微生物除臭方面的研究进展,总结来看,大致分为两方面:第一方面是研究开发新型吸附载体材料,通过改进制备方法,提升材料吸收功能与负载能力,同时降低材料制造成本,为工业化生产提供基础;第二方面是筛选和驯化高效除臭保氮性能的微生物,提高臭气的去除效率及微生物的环境耐受性,以满足生产生活中各类污染气体治理的特异性需求,实现恶臭气体的有效处理。
发明内容
针对现有臭气污染治理技术存在的不足,本发明的目的在于提供一种粪产碱杆菌菌株,具有高效的除臭保氮性能。
本发明的另一目的还在于提供一种炭基固定化菌剂及其制备方法,以及该固定化菌剂在除臭中的应用。
为实现上述发明目的,本发明采用如下技术方案:
一种粪产碱杆菌菌株,其特征在于:分类命名为Alcaligenes faecalis LYC,已于2020年12月28日保藏于中国微生物菌种保藏委员会普通微生物中心(CGMCC),保藏编号为:CGMCC No.21558,保藏地址为:中国,北京,中国科学院微生物研究所。
所述的粪产碱杆菌菌株,其16S rDNA的核苷酸序列如表1所示。
本发明所述的Alcaligenes faecalis LYC的筛选方法如下:
1、首先取新鲜的堆肥腐熟样品在生理盐水中振荡,取10mL上清液加入到90mL的LB培养基中,30℃,150r/min培养3-4d。
2、将富集了3-4d的培养基按照10%的接种量接种于新的100mL以氯化铵为唯一氮源的筛选培养基中,30℃,150r/min再次培养2-3d,如此重复3-5次。
3、将富集初筛完成的培养基稀释涂布在固体筛选培养基上,30℃培养箱中培养,培养4d后,挑选优势成型菌落,平板反复画线分离,直至得到纯种菌Alcaligenes faecalisLYZ。
具体地,所述的筛选培养基为:NH4Cl 0.764g(NH4 +-N 200mg/L),乙酸钠2g,MgSO4·7H2O 0.021g,K2HPO4·3H2O 0.22g,NaHCO3 2.40g,CaCO3 0.05g,FeSO4·7H2O0.073g,MnSO4·4H2O 0.075g,H2O 1000mL。pH值为7.0-7.6,DO控制在2.0-2.5mg/L。固体培养基加入琼脂15~20g/L、121℃灭菌20min。
本发明所述的Alcaligenes faecalis LYC的鉴定方法:
Alcaligenes faecalis LYC是一种异养硝化菌,在硝化菌株分离培养基中长势良好。该菌株平板培养基24h后长出淡黄色菌落,为好氧革兰氏阴性菌,菌株肉眼可看到清晰的单菌落,菌落较小且扁平,呈现不规则圆形,直径0.5-1.0mm左右,湿润。(图1)。Biolog微生物自动分析系统对Alcaligenes faecalis LYC的94种生化表型进行测试,并鉴定菌种。在94个表型测试中分析了71个碳源利用测定和23个化学敏感性测定。由微量分析系统GENIII Micro Plate Biolog确定重要的生物化学特征如表2所示。其中Alcaligenesfaecalis LYC菌株可有效利用18种碳源,包括D-丙氨酸、L-天冬氨酸、L-谷氨酸、L-组氨酸和L-焦谷氨酸等,同时化学敏感性测试中,对13种化学物质敏感,其中包括2种酸性应激物(夫西地酸和pH6),4种渗透应激物(1%NaCl,4%NaCl,8%NaCl和D-丝氨酸),4种抑制剂(1%乳酸钠、二甲胺四环素、硫酸四癸钠和丁酸钠),以及3种毒素(利福霉素,林肯霉素和万古霉素)。菌株LYC生理生化鉴定的结果与粪产碱杆菌属的特征最为接近(Neighbor-Joining法构建系统的进化树如图2)。
所述的异养硝化菌株Alcaligenes faecalis LYC分子水平上的鉴定方法:利用通用引物27F和1492R扩增菌株LYC的16S rDNA部分序列(表1)。将所获得的片段进行测序,NCBI数据库比对该16S rDNA序列,在分子水平上将菌株LYZ鉴定为粪产碱杆菌属。
基于所述的Alcaligenes faecalis LYC,本发明还涉及一种炭基固定化菌剂,其特征在于,包含粪产碱杆菌Alcaligenes faecalis LYC和固定化载体,所述固定化载体为生物炭。
所述炭基固定化菌剂中,所述Alcaligenes faecalis LYC的活菌含量为1*108cfu/g以上。
进一步地,所述固定化载体中还可以添加麦麸,麦麸和生物炭的质量比为1:2-5。
所述炭基固定化菌剂制备的方法,包括如下步骤:
1、固定化载体中加入适量营养液制成吸附剂;
2、将粪产碱杆菌Alcaligenes faecalis LYC培养液加入所述的吸附剂中;
3、将得到的混合物置于恒温培养箱中,在45℃恒温条件下充分搅拌,培养16-20h后固液分离,烘干,保持其中的活菌含量为108cfu/g以上,即得到所述的固定化菌剂。
进一步地,所述生物炭由木质生物质锻烧制得。如具体实施方式中,将秸秆等木质生物质晒干并放入105℃烘箱烘至恒重,粉碎过80目筛;将秸秆粉末与KOH混合,烘干后放入管式炉,在500-700℃下高温绝氧锻烧制备成生物炭。
优选地,所述的营养液的组分为牛肉膏0.5%、蛋白胨1%、氯化钠0.5%、氯化钙0.1%,其余为水,pH为7.0-7.5。
优选地,所述培养液与吸附剂的用量为,培养液:吸附剂为1:1L/kg。
所述炭基固定化菌剂具有除臭保氮性能,将微生物Alcaligenes faecalis LYC与生物炭载体材料搭配,提高臭气的去除效率及微生物的环境耐受性。
因此,本发明还涉及所述的炭基固定化菌剂在臭气治理中的应用。
将所述的炭基固定化菌剂混入,或制成除臭菌液喷洒在产生臭气的堆肥、垃圾、污泥或污水等污染物或废弃物中,能有效吸附脱除恶臭气体,同时固定堆体或污水内的含氮类致臭元素。
有益效果:本发明的粪产碱杆菌Alcaligenes faecalis LYC菌株,具有高效的除臭保氮性能。将所述的微生物菌株负载于生物炭上制成炭基固定化菌剂,既扩大了微生物的接触面积,提高了菌剂对气体的吸附脱除效率,同时生物炭还能减少高温对菌体的胁迫。利用所述的炭基固定化菌剂,可有效吸附恶臭气体于生物炭材料表面,利用微生物固定氮素,进而降低臭气排放浓度。本发明克服了传统的微生物菌剂存在的数量少、活性低、稳定性差及不耐环境冲击的缺点,同时实现了废弃物的资源化利用,解决了树木枝条等木质生物质难于降解、再利用的问题。
附图说明
图1菌株Alcaligenes faecalis LYZ培养的表观图(A)及革兰氏染色图(B);
图2菌株Alcaligenes faecalis LYZ的进化树;
图3Alcaligenes faecalis LYC菌株培养过程中氮素变化的定量分析;
图4炭基固定化菌剂成品图;
图5堆肥过程中氮素变化图,(A)氨气排放、(B)总氮含量、(C)硝氮含量,图中EG表示试验组,CK表示对照组。
具体实施方式
下面结合具体实施例对本发明进行详细描述。本发明的保护范围并不以具体实施方式为限,而是由权利要求加以限定。
以下实施例中所使用的实验方法无特殊说明均为常规方法。所使用的实验试剂耗材等无特殊说明均可从商业途径购买。
实施例1
异养硝化微生物Alcaligenes faecalis LYC的分离筛选:
1、首先取新鲜的堆肥腐熟样品在生理盐水中振荡,取10mL上清液加入到90mL的LB培养基中,30℃,150r/min培养3-4d。
2、将富集了3-4d的培养基按照10%的接种量接种于新的100mL以氯化铵为唯一氮源的筛选培养基中,30℃,150r/min再次培养2-3d,如此重复3-5次。
3、将富集初筛完成的培养基稀释涂布在固体筛选培养基上,30℃培养箱中培养,培养4d后,挑选优势成型菌落,平板反复画线分离,直至得到纯种菌LYZ。
4、对菌株LYZ进行硝化性能分析,氮素变化的定性分析中,二苯胺溶液呈蓝色,格里斯溶液呈红色或褐色,可知该菌株拥有较好的NO3 --N积累能力,如图3所示的定量分析可以发现,菌株LYZ在底物NH4Cl初始浓度为200mg/L时,摇床培养70d后,总氮损失15.8%,硝氮累积49.65%,氨氮降解68.3%。
具体地,所述的筛选培养基为:NH4Cl 0.764g(NH4 +-N 200mg/L),乙酸钠2g,MgSO4·7H2O 0.021g,K2HPO4·3H2O 0.22g,NaHCO3 2.40g,CaCO3 0.05g,FeSO4·7H2O0.073g,MnSO4·4H2O 0.075g,H2O 1000mL。pH值为7.0-7.6,DO控制在2.0-2.5mg/L。固体培养基加入琼脂15~20g/L、121℃灭菌20min。
实施例2
炭基固定化菌剂及其制备方法:
1、取一定数量的木质生物质(秸秆)晒干并放入105℃烘箱烘至恒重,粉碎过80目筛。
2、取所述秸秆粉末与KOH同重量比例加水常温混合,抽滤,烘干,放入管式炉在500-700℃下高温绝氧锻烧制备成生物炭。
3、将麦麸和所述生物炭按1:2重量比例均匀混合,并加入适量营养液制成吸附剂,所述的营养液的组分为牛肉膏0.5%、蛋白胨1%、氯化钠0.5%、氯化钙0.1%,其余为水,pH为7.0-7.5。
4、每升粪产碱杆菌Alcaligenes faecalis LYC培养液加入吸附剂1kg。
5、将混合物置于恒温摇床培养箱中,在45℃恒温条件下充分搅拌,培养16-20h后固液分离,取固相组分于65℃烘干3h,干燥后即得炭基固定化菌剂,保持菌剂中的活菌含量为108cfu/g以上(图4)。
实施例3
炭基固定化菌剂在堆肥过程中对氨气的去除及氮素的保存:
将实施例2制备的炭基固定化菌剂按比例投入堆肥实验,实验采用的堆肥物料为140kg鸡粪鲜样与70kg粉碎后的风干玉米秸秆均匀混合并均分两堆,在实验组(EG)中加入10%的炭基固定化菌剂,以未加入菌剂的堆体为对照处理(CK)。去离子水将两个堆体的含水率调节至65%左右,最后将两堆物料分别置于60L好氧发酵装置中进行发酵实验。实验内2个处理:空白处理(CK):鸡粪+秸秆;对照组处理(EG):鸡粪+秸秆+10%炭基固定化菌剂,两个装置通过气泵连接,通风速度设置为4L/min。结果如图5所示。
从图5可知,在为期30d的鸡粪好氧堆肥实验中,投入10%的炭基固定化菌剂可使堆体排放的氨气浓度相比对照组减少了25.06%,NO3 --N含量相比提高了36.59%,总氮保存率提高了14.6%。
实施例4
将实施例2制备的炭基固定化菌剂与水等重量比例混合制备成除臭菌液,将其洒入垃圾填埋场,研究发现经喷洒菌剂后24h至48h时段内填埋场的臭气浓度出现很大程度的降低,对氨气的去除率维持在65-95%,效果要好于市场上的商品EM菌剂,该菌剂对含氮类臭气的去除效果最为理想。
实施例5
将炭基固定化菌剂与污水按菌液/污水的比值为5/1000的体积比例混合,由于该菌可以利用NH4 +-N作为氮源生长,在投料后的2d内(指数生长期),污水中的NH4 +-N下降了57%。
表1粪产碱杆菌Alcaligenes faecalis LYC的16sDNA核苷酸序列表
ACCTTCTCCATGCAGTTCGAACGGCAGCGCGAGAGAGCTTGCTTCTCCTTTGGCGGCGAGTGGCGGACGGGTGAGTTAATATATCGGAACGTGCCCAGTAGCGGGGGATAACTACTCGAAAGAGTGGCTAATACCGCATACGCCCTACGGGGGAAAGGGGGGGATCGCAAGACCTCTCACTATTGGAGCGGCCGATATCGGATTAGCTAGTTGGTGGGGTAAAGGCTCACCAAGGCAACGATCCGTAGCTGGTTTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGAAACCCTGATCCAGCCATCCCGCGTGTATGATGAAGGCCTTCGGGTTGTAAAGTACTTTTGGCAGAGAAGAAAAGGTATCCCCTAATACGGGATACTGCTGACGGTATCTGCAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGTGTAGGCGGTTCGGAAAGAAAGATGTGAAATCCCAGGGCTCAACCTTGGAACTGCATTTTTAACTGCCGAGCTAGAGTATGTCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGATATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGATAATACTGACGCTCAGACACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGCTGTTGGGGCCGTTAGGCCTTAGTAGCGCAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTCTGGAAAGCCGAAGAGATTTGGCCGTGCTCGCAAGAGAACCGGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACGCAAGAGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGGACAGAGGGTCGCCAACCCGCGAGGGGGAGCCAATCTCAGAAACCCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACAACACCGCCCGTCAACACCAATGGGAAGTGGGTTTCAACCAGGAAAGTAGGTAGCCTAAACCGGAAAGGAGGGCGCTTACCAACGT
表2菌株Alcaligenes faecalis LYZ的生理生化特征测定
序列表
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accttctcca tgcagttcga acggcagcgc gagagagctt gcttctcctt tggcggcgag 60
tggcggacgg gtgagttaat atatcggaac gtgcccagta gcgggggata actactcgaa 120
agagtggcta ataccgcata cgccctacgg gggaaagggg gggatcgcaa gacctctcac 180
tattggagcg gccgatatcg gattagctag ttggtggggt aaaggctcac caaggcaacg 240
atccgtagct ggtttgagag gacgaccagc cacactggga ctgagacacg gcccagactc 300
ctacgggagg cagcagtggg gaattttgga caatggggga aaccctgatc cagccatccc 360
gcgtgtatga tgaaggcctt cgggttgtaa agtacttttg gcagagaaga aaaggtatcc 420
cctaatacgg gatactgctg acggtatctg cagaataagc accggctaac tacgtgccag 480
cagccgcggt aatacgtagg gtgcaagcgt taatcggaat tactgggcgt aaagcgtgtg 540
taggcggttc ggaaagaaag atgtgaaatc ccagggctca accttggaac tgcattttta 600
actgccgagc tagagtatgt cagagggggg tagaattcca cgtgtagcag tgaaatgcgt 660
agatatgtgg aggaataccg atggcgaagg cagccccctg ggataatact gacgctcaga 720
cacgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc ctaaacgatg 780
tcaactagct gttggggccg ttaggcctta gtagcgcagc taacgcgtga agttgaccgc 840
ctggggagta cggtcgcaag attaaaactc aaaggaattg acggggaccc gcacaagcgg 900
tggatgatgt ggattaattc gatgcaacgc gaaaaacctt acctaccctt gacatgtctg 960
gaaagccgaa gagatttggc cgtgctcgca agagaaccgg aacacaggtg ctgcatggct 1020
gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtca 1080
ttagttgcta cgcaagagca ctctaatgag actgccggtg acaaaccgga ggaaggtggg 1140
gatgacgtca agtcctcatg gcccttatgg gtagggcttc acacgtcata caatggtcgg 1200
gacagagggt cgccaacccg cgagggggag ccaatctcag aaacccgatc gtagtccgga 1260
tcgcagtctg caactcgact gcgtgaagtc ggaatcgcta gtaatcgcgg atcagaatgt 1320
cgcggtgaat acgttcccgg gtcttgtaca acaccgcccg tcaacaccaa tgggaagtgg 1380
gtttcaacca ggaaagtagg tagcctaaac cggaaaggag ggcgcttacc aacgt 1435
Claims (10)
1.一种粪产碱杆菌菌株,其特征在于:分类命名为Alcaligenes faecalis LYC,保藏编号为:CGMCC No.21558。
2.根据权利要求1所述的粪产碱杆菌菌株,其特征在于:其16S rDNA的核苷酸序列如表1所示。
3.一种炭基固定化菌剂,其特征在于,包含粪产碱杆菌Alcaligenes faecalis LYC和固定化载体,所述固定化载体为生物炭。
4.根据权利要求3所述的炭基固定化菌剂,其特征在于,所述Alcaligenes faecalisLYC的活菌含量为1*108cfu/g以上。
5.根据权利要求3所述的炭基固定化菌剂,其特征在于,所述固定化载体中还添加麦麸,麦麸和生物炭的质量比为1:2-5。
6.权利要求3所述的炭基固定化菌剂的制备方法,包括以下步骤:
1)固定化载体中加入适量营养液制成吸附剂;
2)将粪产碱杆菌Alcaligenes faecalis LYC培养液加入所述的吸附剂中;
3)将得到的混合物置于恒温培养箱中,在45℃恒温条件下充分搅拌,培养16-20h后固液分离,烘干,保持其中的活菌含量为108cfu/g以上,得到所述的固定化菌剂。
7.根据权利要求6所述的炭基固定化菌剂的制备方法,其特征在于,所述的营养液的组分为牛肉膏0.5%、蛋白胨1%、氯化钠0.5%、氯化钙0.1%,其余为水,pH为7.0-7.5。
8.根据权利要求6所述的炭基固定化菌剂的制备方法,其特征在于,所述培养液与吸附剂的用量为培养液:吸附剂为1:1L/kg。
9.权利要求6所述的炭基固定化菌剂在臭气治理中的应用。
10.根据权利要求9所述的应用,其特征在于,将所述的炭基固定化菌剂混入,或制成除臭菌液喷洒在产生臭气的污染物或废弃物中。
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CN114958686A (zh) * | 2022-06-24 | 2022-08-30 | 浙江省农业科学院 | 一种醌基修饰生物炭基微生物菌剂及其制备和应用 |
CN114958686B (zh) * | 2022-06-24 | 2023-08-08 | 浙江省农业科学院 | 一种醌基修饰生物炭基微生物菌剂及其制备和应用 |
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