短期生物炭添加对不同类型土壤细菌和氨氧化微生物的影响

doi:10.3864/j.issn.0578-1752.2019.07.013

摘要/Abstract

摘要：

【目的】氨氧化作用是硝化作用的第一步,也是硝化作用的限速步骤,是全球氮循环的关键环节。本试验旨在研究在我国不同类型土壤中添加花生壳生物炭对细菌和氨氧化作用的影响,为生物炭的推广使用提供理论依据。【方法】试验以黄棕壤、潮土、黑土为供试土壤,通过短期培养试验,利用16SrRNA测序研究生物炭对不同类型土壤氨氧化微生物、细菌群落结构以及相关酶基因表达量的影响。每种土壤设置4个处理：CK（不施用化肥和生物炭）,F（单施化肥）,C（单施2%花生壳生物炭）,FC（施用化肥+2%花生壳生物炭）。【结果】施用生物炭后（C、FC）酸性土壤pH显著提高了0.5—1.0个单位,但碱性土壤pH显著降低了0.5—0.6个单位;单施生物炭（C）造成黄棕壤的微生物丰富度和多样性显著提高,潮土在单施生物炭（C）时仅显著提高了土壤的微生物多样性指数,在黑土中施用生物炭和化肥都未显著改变土壤微生物的丰富度和多样性;在3种土壤中氨氧化细菌的丰度皆高于氨氧化古菌,测得的氨氧化细菌的OTU丰度约为氨氧化古菌的8.1倍;生物炭和化肥并未显著改变奇古菌门中的OTU丰度,却对β和γ变形菌中的OTU丰度产生了显著性影响;3种土壤的氨氧化细菌都以β变形菌为主,约占60%;另外,生物炭的施用（C、FC）在PC1（40.4%）上显著改变了黄棕壤的微生物群落结构,在PC1（42.3%）和PC2（21.3%）上都显著改变了潮土的微生物群落结构;施用生物炭后（C、FC）,短期内潮土中氨合成相关酶基因表达量显著降低14.7%—39.9%,氨氧化古菌丰度在单一施炭（C）和化肥与生物炭同施（FC）时分别降低了70.5%和48.7%。【结论】施用生物炭后,短期内显著改变了黄棕壤和潮土的微生物群落结构,并明显抑制了潮土的氨氧化作用。

关键词: 生物炭, 化肥, 氨氧化作用, 微生物群落结构, 黄棕壤, 潮土, 黑土

Abstract:

【Objective】Ammonia oxidation is the first step in nitrification and the rate-limiting step in nitrification. It is a key link in the global nitrogen cycle. The purpose of this experiment was to study the effects of peanut shell biochar application on bacteria and ammonia oxidation in different type soils in China, so as to provide a theoretical basis for the promotion and use of biochar.【Method】Yellow-brown soil, fluvo-aquic soil and black soil were utilized as the tested soil. Through short-term culture experiments, 16SrRNA sequencing was used to study the effects of biochar on ammonia-oxidizing microorganisms, bacterial community structure and related enzyme gene expression in different type soils. Four treatments for each soil included CK (no fertilizer and biochar), F (single fertilization), C (single 2% peanut shell biochar), and FC (application of fertilizer + 2% peanut shell biochar).【Result】Acid soil pH increased significantly by 0.5-1.0 units after application of biochar (C, FC), but alkaline soil pH decreased significantly by 0.5-0.6 units; the microbial abundance and diversity of yellow-brown soil increased significantly caused by single application of biochar (C). The fluvo-aquic only significantly increased the microbial diversity index of the soil when it was applied to biochar alone (C). Biochar and chemical fertilizers did not significantly change soil abundance and diversity in black soil; the abundance of ammonia-oxidizing bacteria in three soils was higher than that of ammonia-oxidizing archaea. The measured OTU abundance of ammonia-oxidizing bacteria was about 8.1 times that of ammonia-oxidizing archaea. Biochar and chemical fertilizers did not significantly alter the OTU abundance in the thaumarchaeota, but had a significant effect on the OTU abundance in the beta and gamma proteobacteria. The ammonia-oxidizing bacteria of the three soils were mainly β-proteobacteria, accounting for about 60%. In addition, the application of biochar (C, FC) significantly changed the microbial community structure of yellow-brown soil on PC1 (40.4%), and significantly changed the microbial community structure of fluvo-aquic soil on both PC1 (42.3%) and PC2 (21.3%). After application of biochar (C, FC), the expression of ammonia synthesis related enzyme gene in fluvo-aquic soil decreased significantly by 14.7%-39.9% in a short period of time, and the ammoxidation archaea abundance decreased by 70.5% and 48.7% under C treatment and FC treatment, respectively.【Conclusion】After application of biochar, the microbial community structure of yellow-brown soil and fluvo-aquic soil was significantly changed, and the ammoxidation of fluvo-aquic soil was obviously inhibited in a short period of time.

Key words: biochar, chemical fertilizer, ammonia oxidation, microbial community structure, yellow-brown soil, fluvo-aquic soil, black soil

张梦阳,夏浩,吕波,丛铭,宋文群,姜存仓. 短期生物炭添加对不同类型土壤细菌和氨氧化微生物的影响[J]. 中国农业科学, 2019, 52(7): 1260-1271.

ZHANG MengYang,XIA Hao,LÜ Bo,CONG Ming,SONG WenQun,JIANG CunCang. Short-Term Effect of Biochar Amendments on Total Bacteria and Ammonia Oxidizers Communities in Different Type Soils[J]. Scientia Agricultura Sinica, 2019, 52(7): 1260-1271.

图/表 10

表1

图1

表2

图2

表3

图3

图4

图5

表4

表5

参考文献 51

[1]	CANFIELD D E, GLAZER A N, FALKOWSKI P G . The evolution and future of Earth's nitrogen cycle. Science, 2010,330(6001):192-196. doi: 10.1126/science.1186120 pmid: 20929768
[2]	李霞 . 微生物在氮循环中的作用. 松辽学刊(自然科学版), 1998,10(4):30-33.
	LI X . The role of microorganisms in the nitrogen cycle. Songliao Journal (Natural Science Edition), 1998,10(4):30-33. (in Chinese)
[3]	贺纪正, 张丽梅 . 氨氧化微生物生态学与氮循环研究进展. 生态学报, 2009,29(1):406-415. doi: 10.3321/j.issn:1000-0933.2009.01.049
	HE J Z, ZHANG L M . Advances in ammonia oxidation microbial ecology and nitrogen cycle. Journal of Ecology, 2009,29(1):406-415. (in Chinese) doi: 10.3321/j.issn:1000-0933.2009.01.049
[4]	陈温福, 张伟明, 孟军, 徐正进 . 生物炭应用技术研究. 中国工程科学, 2011,13(2):83-89. doi: 10.3969/j.issn.1009-1742.2011.02.015
	CHEN W F, ZHANG W M, MENG J, XU Z J . Biochar application technology research. Chinese Engineering Science, 2011,13(2):83-89. (in Chinese) doi: 10.3969/j.issn.1009-1742.2011.02.015
[5]	陈心想, 何绪生, 耿增超, 张雯, 高海英 . 生物炭对不同土壤化学性质、小麦和糜子产量的影响. 生态学报, 2013,33(20):6534-6542. doi: 10.5846/stxb201212281891
	CHEN X X, HE X S, GENG Z C, ZHANG W, GAO H Y . Effects of biochar on different soil chemical properties, wheat and hazelnut yield. Journal of Ecology, 2013,33(20):6534-6542. (in Chinese) doi: 10.5846/stxb201212281891
[6]	郑瑞伦, 王宁宁, 孙国新, 谢祖彬, 庞卓, 王庆海, 武菊英 . 生物炭对京郊沙化地土壤性质和苜蓿生长、养分吸收的影响. 农业环境科学学报, 2015,34(5):904-912. doi: 10.11654/jaes.2015.05.013
	ZHENG R L, WANG N N, SUN G X, XIE Z B, PANG Z, WANG Q H, WU J Y . Effects of biochar on soil properties, alfalfa growth and nutrient uptake in desertification areas of Beijing suburbs. Journal of Agricultural Environmental Science, 2015,34(5):904-912. (in Chinese) doi: 10.11654/jaes.2015.05.013
[7]	李昌见, 屈忠义, 勾芒芒, 苏永莉, 霍星 . 生物炭对土壤水肥利用效率与番茄生长影响研究. 农业环境科学学报, 2014,33(11):2187-2193. doi: 10.11654/jaes.2014.11.017
	LI C J, QU Z Y, GOU M M, SU Y L, HUO X . Effects of biochar on soil water and fertilizer utilization efficiency and tomato growth. Journal of Agricultural Environmental Science, 2014,33(11):2187-2193. (in Chinese) doi: 10.11654/jaes.2014.11.017
[8]	吕波, 王宇函, 夏浩, 姚子涵, 姜存仓 . 不同改良剂对黄棕壤和红壤上白菜生长及土壤肥力影响的差异. 中国农业科学, 2018,51(22):4306-4315.
	LV B, WANG Y H, XIA H, YAO Z H, JIANG C C . Effects of Biochar and Other Amendments on the Cabbage Growth and Soil Fertility in Yellow-Brown Soil. Scientia Agricultura Sinica, 2018,51(22):4306-4315. (in Chinese)
[9]	应介官, 林庆毅, 张梦阳, 黄毅, 彭抒昂, 姜存仓 . 生物炭对铝富集酸性土壤的毒性缓解效应及潜在机制. 中国农业科学, 2016,49(23):4576-4583. doi: 10.3864/j.issn.0578-1752.2016.23.010
	YING J G, LIN Q Y, ZHANG M Y, HUANG Y, PENG S A, JIANG C C . Mitigative effect of biochar on aluminum toxicity of acid soil and the potential mechanism. Scientia Agricultura Sinica, 2016,49(23):4576-4583. (in Chinese) doi: 10.3864/j.issn.0578-1752.2016.23.010
[10]	ASADA T, ISHIHARA S, YAMANE T, TOBA A, YAMADA A . Science of bamboo charcoal: Study of carbonizing temperature of bamboo charcoal and removal capability of harmful gases. Journal of Health Science, 2002,48(6):473-479. doi: 10.1248/jhs.48.473
[11]	ASADA T, OHKUBO T, KAWATA K, OIKAWA K . Ammonia adsorption on bamboo charcoal with acid treatment. Journal of Health Science, 2006,52(5):585-589. doi: 10.1248/jhs.52.585
[12]	CLOUGH T J, BERTRAM J E, RAY J L, CONDRON L M, O'CALLAGHAN M, SHERLOCK R R, WELLS N S . Unweathered wood biochar impact on nitrous oxide emissions from a bovine-urine- amended pasture soil. Soil Science Society of America Journal, 2010,74(3):852-860. doi: 10.2136/sssaj2009.0185
[13]	CAYUELA M L, SÁNCHEZ-MONEDERO M A, ROIG A, HANLEY K, ENDERS A, LEHMANN J . Biochar and denitrification in soils: when, how much and why does biochar reduce N₂O emissions? Scientific Reports, 2013,3:1732. doi: 10.1038/srep01732 pmid: 3635057
[14]	VERHOEVEN E, SIX J . Biochar does not mitigate field-scale N₂O emissions in a Northern California vineyard: An assessment across two years. Agriculture, Ecosystems ＆ Environment, 2014,191(15):27-38. doi: 10.1016/j.agee.2014.03.008
[15]	PURKHOLD U, POMMERENINGRÖSER A, JURETSCHKO S, SCHMID M C, KOOPS H P, WAGNER M . Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys. Applied and Environmental Microbiology, 2000,66(12):5368-5382. doi: 10.1128/AEM.66.12.5368-5382.2000 pmid: 92470
[16]	MONTEIRO M, SÉNECA J, MAGALHÃES C . The history of aerobic ammonia oxidizers: from the first discoveries to today. Journal of Microbiology, 2014,52(7):537-547. doi: 10.1007/s12275-014-4114-0 pmid: 24972807
[17]	TREUSCH A, LEININGER S, KLETZIN A, SCHUSTER S, KLENK H, SCHLEPER C . Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling. Environmental Microbiology, 2005,7(12):1985-1995. doi: 10.1111/j.1462-2920.2005.00906.x pmid: 16309395
[18]	BROCHIERARMANET C, BOUSSAU B, GRIBALDO S, FORTERRE P . Mesophilic crenarchaeota: proposal for a third archaeal phylum, the Thaumarchaeota. Nature Reviews Microbiology, 2008,6(3):245-252. doi: 10.1038/nrmicro1852
[19]	PESTER M, SCHLEPER C, WAGNER M . The Thaumarchaeota: an emerging view of their phylogeny and ecophysiology. Current Opinion in Microbiology, 2011,14(3):300-306. doi: 10.1016/j.mib.2011.04.007 pmid: 21546306
[20]	SINGH B P, HATTON B J, BALWANT S, COWIE A L, KATHURIA A . Influence of biochars on nitrous oxide emission and nitrogen leaching from two contrasting soils. Journal of Environmental Quality, 2010,39(4):1224-1235. doi: 10.2134/jeq2009.0138 pmid: 20830910
[21]	STEINER C, TEIXEIRA W G, LEHMANN J, NEHLS T, DE MACÊDO J L V, BLUM W E H, ZECH W . Long term effects of manure, charcoal and mineral fertilization on crop production and fertility on a highly weathered Central Amazonian upland soil. Plant and Soil, 2007,291(1/2):275-290. doi: 10.1007/s11104-007-9193-9
[22]	RONDON M A, LEHMANN J, RAMÍREZ J, HURTADO M . Biological nitrogen fixation by common beans (Phaseolus vulgaris L.) increases with bio-char additions. Biology and Fertility of Soils, 2007,43(6):699-708. doi: 10.1007/s00374-006-0152-z
[23]	BALL P N, MACKENZIE M D, DELUCA T H, HOLBEN W E . Wildfire and charcoal enhance nitrification and ammonium-oxidizing bacterial abundance in dry montane forest soils. Journal of Environmental Quality, 2010,39(4):1243-1253. doi: 10.2134/jeq2009.0082 pmid: 20830912
[24]	SPOKAS K A, REICOSKY D C . Impacts of sixteen different biochars on soil greenhouse gas production. Annals of Environmental Science, 2009,3(1):179-193.
[25]	ABUJABHAH I S, DOYLE R B, BOUND S A, BOWMAN J P . Assessment of bacterial community composition, methanotrophic and nitrogen-cycling bacteria in three soils with different biochar application rates. Journal of Soils & Sediments, 2018,18(1):148-158. doi: 10.1007/s11368-017-1733-1
[26]	LIU S N, MENG J, JIANG L L, YANG X, LAN Y, CHENG X Y, CHEN W F . Rice husk biochar impacts soil phosphorous availability, phosphatase activities and bacterial community characteristics in three different soil types. Applied Soil Ecology, 2017,116:12-22. doi: 10.1016/j.apsoil.2017.03.020
[27]	MAGOČ T, SALZBERG S L . FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics, 2011,27(22):2957-2963. doi: 10.1093/bioinformatics/btr507
[28]	EDGAR R C . UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature Methods, 2013,10(10):996. doi: 10.1038/NMETH.2604 pmid: 23955772
[29]	WANG Q, GARRITY G M, TIEDJ J M, COLE J R . Naïve bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology, 2007,73(16):5261-5267. doi: 10.1128/AEM.00062-07
[30]	CHAO A, BUNGE J . Estimating the number of species in a stochastic abundance model. Biometrics, 2002,58(3):531-539. doi: 10.1111/j.0006-341X.2002.00531.x pmid: 12229987
[31]	LANGILLE M G I, ZANEVELD J, CAPORASO J G, MCDONALD D, KNIGHTS D, REYES J A, CLEMENTE J C, BURKEPILE D E, THURBER R L V, KNIGHT R, BEIKO R G, HUTTENHOWER C . Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nature Biotechnology, 2013,31(9):814. doi: 10.1038/nbt.2676 pmid: 3819121
[32]	郭赟 . 长期施肥对酸性及中性水稻土壤中氨氧化微生物的影响[D]. 南京: 南京师范大学, 2013.
	GUO Y . Effects of long-term fertilization on ammonia-oxidizing microorganisms in acidic and neutral rice soils[D]. Nanjing: Nanjing Normal University, 2013. ( in Chinese)
[33]	PROSSER J I . Autotrophic nitrification in bacteria. Advances in Microbial Physiology, 1989,30(1):125-181.
[34]	LEININGER S, URICH T, SCHLOTER M, SCHWARK L, QI J, NICOL G W, PROSSER J I, SCHUSTER S C, SCHLEPER C . Archaea predominate among ammonia-oxidizing prokaryotes in soils. Nature, 2006,442:806-809. doi: 10.1038/nature04983 pmid: 16915287
[35]	NICOLE G W, LEININGER S, SCHLEPER C, PROSSER J I . The influence of soil pH on the diversity, abundance and transcriptional activity of ammonia oxidizing archaea and bacteria. Environmental Microbiology, 2010,10(11):2966-2978. doi: 10.1111/j.1462-2920.2008.01701.x pmid: 18707610
[36]	NEJC S, GUBRYRANGIN C, ŠPELA H, GRAEME W N, INES M, JAMES I P . Thaumarchaeal ammonia oxidation in an acidic forest peat soil is not influenced by ammonium amendment. Applied and Environmental Microbiology, 2010,76(22):7626. doi: 10.1128/AEM.00595-10 pmid: 20889787
[37]	LIAO X, CHEN C, ZHANG J, DAI Y, ZHANG X, XIE S . Operational performance, biomass and microbial community structure: impacts of backwashing on drinking water biofilter. Environmental Science and Pollution Research International, 2015,22(1):546. doi: 10.1007/s11356-014-3393-7 pmid: 25087501
[38]	HE L L, BI Y C, ZHAO J, PITTELKOW C M, ZHAO X, WANG S Q, XING G X . Population and community structure shifts of ammonia oxidizers after four-year successive biochar application to agricultural acidic and alkaline soils. Science of the Total Environment, 2018,619:1105-1115. doi: 10.1016/j.scitotenv.2017.11.029 pmid: 29734589
[39]	李双双, 陈晨, 段鹏鹏, 许欣, 熊正琴 . 生物质炭对酸性菜地土壤NO排放及相关功能基因丰度的影响. 植物营养与肥料学报, 2018,24(2):414-423. doi: 10.11674/zwyf.17272
	LI S S, CHEN C, DUAN P P, XU X, XIONG Z Q . Effects of biochar on NO emission and related functional gene abundance in acidic vegetable soils. Journal of Plant Nutrition and Fertilizer, 2018,24(2):414-423. (in Chinese) doi: 10.11674/zwyf.17272
[40]	刘远, 朱继荣, 吴雨晨, 束良佐 . 施用生物质炭对采煤塌陷区土壤氨氧化微生物丰度和群落结构的影响. 应用生态学报, 2017,28(10):3417-3423. doi: 10.13287/j.1001-9332.201710.034
	LIU Y, ZHU J R, WU Y C, SHU L Z . Effects of application of biomass carbon on ammonia microbial abundance and community structure in coal mining subsidence area. Chinese Journal of Applied Ecology, 2017, 28(10):3417-3423. (in Chinese) doi: 10.13287/j.1001-9332.201710.034
[41]	BI Q F, CHEN Q H, YANG X R, LI H, ZHENG B X, ZHOU W W, LIU X X, DAI P B, LI K J, LIN X Y . Effects of combined application of nitrogen fertilizer and biochar on the nitrification and ammonia oxidizers in an intensive vegetable soil. Amb Express, 2017,7:108. doi: 10.1186/s13568-017-0363-8 pmid: 28571306
[42]	WU H P, ZENG G M, LIANG J, CHEN J, XU J J, DAI J, LI X D, CHEN M, XU P, ZHOU Y Y, LI F, HU L, WAN J . Responses of bacterial community and functional marker genes of nitrogen cycling to biochar, compost and combined amendments in soil. Environmental Biotechnology, 2016, 100:8583-8591. doi: 10.1007/s00253-016-7614-5 pmid: 27338575
[43]	JIN H . Thesis: Characterization of microbial life colonizing biochar and biochar-amended soils[D]. Ithaca: Cornell University, 2010.
[44]	KHODADAD C L M, ZIMMERMAN A R, GREEN S J, UTHANDI S, FOSTER J S . Taxa-specific changes in soil microbial community composition induced by pyrogenic carbon amendments. Soil Biology & Biochemistry, 2011,43(2):385-392. doi: 10.1016/j.soilbio.2010.11.005
[45]	ALLISON S D, MARTINY J B H . Resistance, resilience, and redundancy in microbial communities. Proceedings of the National Academy of Sciences of the United States of America, 2008,105(32):11512-11519. doi: 10.1073/pnas.0801925105
[46]	BÖRJESSON G, MENICHETTI L, KIRCHMANN H, KATTERER T . Soil microbial community structure affected by 53 years of nitrogen fertilisation and different organic amendments. Biology & Fertility of Soils, 2012,48(3):245-257. doi: 10.1007/s00374-011-0623-8
[47]	HALLIN S, JONES C M, SCHLOTER M, PHILIPPOT L . Relationship between N-cycling communities and ecosystem functioning in a 50-year-old fertilization experiment. Isme Journal, 2009,3(5):597. doi: 10.1038/ismej.2008.128 pmid: 19148144
[48]	HARTMANN M, FLIESSBACH A, OBERHOLZER H R, WIDMER F . Ranking the magnitude of crop and farming system effects on soil microbial biomass and genetic structure of bacterial communities. FEMS Microbiology Ecology, 2006,57(3):378-388. doi: 10.1111/j.1574-6941.2006.00132.x pmid: 16907752
[49]	ZHONG W H, GU T, WEI W, ZHANG B, LIN X, HUANG Q, SHEN W . The effects of mineral fertilizer and organic manure on soil microbial community and diversity. Plant and Soil, 2010,326(1/2):511-522. doi: 10.1007/s11104-009-9988-y
[50]	GEISSELER D, LAZICKI P A, SCOW K M . Mineral nitrogen input decreases microbial biomass in soils under grasslands but not annual crops. Applied Soil Ecology, 2016,106:1-10. doi: 10.1016/j.apsoil.2016.04.015
[51]	OGILVIE L A, HIRSCH P R, JOHNSTON A W B . Bacterial diversity of the Broadbalk ‘Classical’ winter wheat experiment in relation to long-term fertilizer inputs. Microbial Ecology, 2008,56(3):525-537. doi: 10.1007/s00248-008-9372-0 pmid: 18347845

土壤类型 Soil type	质地 Soil texture	pH	碱解氮 Available nitrogen (mg·kg^-1)	速效磷 Available phosphorus (mg·kg^-1)	速效钾 Available potassium (mg·kg^-1)	有机质 Organic matter (g·kg^-1)
黄棕壤Yellow-brown soil (YB)	黏土Clay	5.2	77.7	49.2	169.4	13.3
潮土Fluvo-aquic soil (FA)	壤土Loam	5.6	43.9	9.3	84.7	8.7
黑土Black soil (B)	黏土Clay	8.8	125.5	3.6	259.2	32.5

因素Factor	F	P value
单施化肥处理F treatment	38.379	0.000
单施生物炭处理C treatment	271.157	0.000
土壤类型Soil type	4304.294	0.000
F×C	11.815	0.002
F×Soil type	57.887	0.000
C×Soil type	121.239	0.000
F×C×Soil type	0.204	0.817

处理 Treatment	奇古菌Thaumarchaeota			β和γ变形菌β, γ- Proteobacteria
处理 Treatment	YB	FA	B	YB	FA	B
CK	152a	216a	763a	4768ab	6156ab	2141ab
F	285a	194a	873a	4744ab	5726b	1937b
C	219a	63a	1259a	3997b	6626a	1918b
FC	130a	110a	1121a	5267a	6417a	2969a

土壤类型Soil type	CK	F	C	FC
YB	260856a	250203a	238879a	230877a
FA	263525a	253256ab	242993bc	234804c
B	225273a	220124a	223533a	226165a

酶 Enzyme	EC（编号）EC (number)	YB				FA				B
酶 Enzyme	EC（编号）EC (number)	CK	F	C	FC	CK	F	C	FC	CK	F	C	FC
甲酰胺酶 Formamidase	1.7.2.1	1073a	1384a	1014a	1083a	1077a	1069a	892a	933a	361b	328b	329b	477a
腈水解酶 Nitrilase	3.5.1.49	4513ab	5000a	4044b	4582ab	5402a	6062a	5352a	5025a	5849a	5448a	5232a	6008a
氰酸裂合酶 Cyanate lyase	3.5.5.1	7036ab	7303ab	5903b	7985a	9535a	8958a	9566a	9398a	9240ab	8538bc	8168c	9710a
氨基甲酸酯激酶 Carbamate kinase	4.2.1.104	4006ab	3632b	3521b	4448a	4539a	4761a	4592a	4837a	4657a	4547a	4384a	4533a
谷氨酸脱氢酶 Glutamate dehydrogenase	2.7.2.2	4984ab	5332a	4574ab	4200b	5148a	5068a	3263b	3092b	9561a	9745a	10561a	10095a
谷氨酸脱氢酶(NAD(P)+) Glutamate dehydrogenase ((NAD(P)+)	1.4.1.2	9631ab	10188a	8444b	9389ab	10659a	11757a	8684b	8277b	17594a	16898a	16405a	16705a
谷氨酸脱氢酶(NADP+) Glutamate dehydrogenase (NADP+)	1.4.1.3	43350a	42063a	41901a	35676a	43520a	39070b	36778b	37143b	29553a	32706a	35500a	32690a
亚硝酸还原酶 (不分解) Nitrite reductase (No-forming)	1.4.1.4	13424a	11436a	10945a	12922a	12085bc	14166a	13367ab	10763c	13604a	13240a	14048a	13256a