Long-term fertilization effects on active ammonia oxidizers in an acidic upland soil in China

The effects of long-term fertilization of acidic soils on ammonia-oxidizing archaea (AOA) and bacteria (AOB) communities and its ecological implications remain poorly understood. We chose an acidic upland soil site under long-term (27-year) fertilization to investigate ammonia oxidizer communities under four different regimes: mineral N fertilizer (N), mineral NPK fertilizer (NPK), organic manure (OM) and an unfertilized control (CK). Soil net nitrification rates were significantly higher in OM soils than in CK, N or NPK soils. Quantitative analysis of the distribution of amoA genes by DNA-based stable isotope probing revealed that AOA dominate in CK, N and NPK soils, while AOB dominate in OM soils. Denaturing gradient gel electrophoresis and clone library analyses of amoA genes revealed that Group 1.1a-associated AOA (also referred to as Nitrosotalea) were the most dominant active AOA population (>92%), while Nitrosospira Cluster 3 and Cluster 9 were predominant among active AOB communities. The functional diversity of active ammonia oxidizers in acidic soils is affected by long-term fertilization practices, and the responses of active ammonia oxidizers to mineral fertilizer and organic manure are clearly different. Our results provide strong evidence that AOA are more highly adapted to growth at low pH and low substrate availability than AOB, and they suggest that the niche differentiation and metabolic diversity of ammonia oxidizers in acidic soils are more complex than previously thought.     

不同施肥方式对红壤蔬菜田氨氧化细菌和氨氧化古菌群落的影响

通过构建氨单加氧酶基因(amoA)克隆文库,研究在红壤蔬菜田上只施用磷钾化肥(PK)、只施氮磷钾化肥(NPK)、施用腐熟有机肥(DNPK)和施用新鲜有机肥(FNPK)等4种不同施肥处理的土壤氨氧化细菌(AOB)和氨氧化古菌(AOA)群落多样性及与土壤脲酶活性的相关性。结果表明:施加有机肥处理(DNPK和FNPK)的蔬菜田土壤的AOB文库和AOA文库OTU数量和Shannon指数高于只施用无机肥(NPK和PK)处理的蔬菜田土壤;DNPK和FNPK处理的土壤优势AOB菌群为多形亚硝化叶菌(Nitrosolobus multiformis),比例分别为88.5%和68.5%,NPK和PK处理的土壤优势AOB菌群为亚硝化单胞菌属(Nitrosospirasp.),比例分别为54.8%和65.5%;DNPK、FNPK、NPK和PK处理土壤优势AOA菌群均为阿伯丁土壤亚硝化细杆菌侯选种(CandidatusNitrosotalea devanaterra),比例分别为90.9%、84.4%、77.8%和45.2%;施加有机肥处理(DNPK和FNPK)的土壤脲酶活性和氨氧化微生物的多样性指数都高于只施用无机肥处理(NPK和PK);AOA群落多样性指数与土壤脲酶活性呈显著正相关,而AOB群落多样性与土壤脲酶活性相关性不显著。总体来看,有机肥比无机肥处理提高了AOA和AOB群落多样性,且AOA在红壤蔬菜田土壤氨氧化过程中起着更为重要的作用。     

Cashew nut shell liquid potentially mitigates methane emission from the feces of Thai native ruminant livestock by modifying fecal microbiota

The methane-mitigating potency of cashew nutshell liquid (CNSL) was evaluated by investigating gas production from batch cultures using feces from Thai native ruminants that had been incubated for different periods. Feces was obtained from four Thai native cattle and four swamp buffaloes reared under practical feeding conditions at the Kasetsart University farm, Thailand. Fecal slurry from the same farm was also included in the analysis. CNSL addition successfully suppressed the methane production potential of feces from both ruminants by shifting short chain fatty acid profiles towards propionate production. Methane mitigation continued for almost 150 days, although the degree of mitigation was more apparent from Day 0 to Day 30. Bacterial and archaeal community shifts with CNSL addition were observed in feces from both ruminants; specifically, Bacteroides increased, whereas Lachnospiraceae and Ruminococcaceae decreased in feces to which CNSL was added. Fecal slurry did not show marked changes in gas production with CNSL addition. The findings showed that the addition of CNSL to the feces of ruminants native to the Southeast Asian region can suppress methane emission. Because CNSL can be easily obtained as a byproduct of the local cashew industry in this region, its on-site application might be ideal.     

Growth and morphologic response of rumen methanogenic archaea and bacteria to cashew nut shell liquid and its alkylphenol components

The growth and morphology of rumen methanogenic archaea (15 strains of 10 species in 5 genera, including 7 strains newly isolated in the present study) and bacteria (14 species in 12 genera) were investigated using unsupplemented in vitro pure cultures and cultures supplemented with cashew nut shell liquid (CNSL) and its phenolic compound components, anti-methanogenic agents for ruminant animals. Growth of most of the methanogens tested was inhibited by CNSL and alkylphenols at different concentrations ranging from 1.56 to 12.5 μg/ml. Of the alkylphenols tested, anacardic acid exhibited the most potent growth inhibition. Three gram-negative bacterial species involved in propionate production were resistant to CNSL and alkylphenols (>50 μg/ml). All the methanogens and bacteria that were sensitive to CNSL and alkylphenols exhibited altered morphology; disruption of the cell surface was notable, possibly due to surfactant activity of the tested materials. Cells division was inhibited in some organisms, with cell elongation and unclear septum formation observed. These results indicate that CNSL and alkylphenols, particularly anacardic acid, inhibit both rumen bacteria and methanogens in a selective manner, which could help mitigate rumen methane generation.     

Digestive physiology,metabolism and methane production of captive Linné's two‐toed sloths (Choloepus didactylus)

Sloths are renowned for their low metabolic rate, low food intake and low defecation frequency. We investigated factors of digestive physiology and energy metabolism in four captive individuals (mean body mass 10.0 ± SD 3.7 kg) of a hitherto mostly unstudied sloth species, Linné's two‐toed sloth (Choloepus didactylus), in a 2‐week digestion recording and 23‐h respiration experiment on animals fed a standard zoo diet of vegetables and starchy components. Dry matter intake, defecation frequency and particle mean retention time (MRT) in the gastrointestinal tract (GIT) were 12 ± 3 g/(kg^0.75 day), once every 5 days and >140 h in three individuals, but 53 g/(kg^0.75 day), daily and 82 h in one individual that was apparently compensating for a period of weight loss prior to the experiment. In all animals, solute marker was eliminated at a faster rate than the particle marker, indicating ‘digesta washing’ in the sloths’ GIT. The overall metabolic rate calculated from oxygen consumption matched the metabolisable energy intake in three individuals [173 ± 22 vs. 168 ± 44 kJ/(kg^0.75 day)] but not in the fourth one [225 vs. 698 kJ/(kg^0.75 day)], supporting the interpretation that this animal was replenishing body stores. In spite of the low food intake and the low‐fibre diet (209 ± 26 g neutral detergent fibre/kg dry matter), methane production was rather high accounting for 9.4 ± 0.8% of gross energy intake (2.7% in the fourth individual), which exceeded literature data for ruminants on forage‐only diets. These results corroborate literature reports on low intake, low defecation frequency, low metabolic rate and long MRT in other sloth species. The long MRT is probably responsible for the comparatively high methane production, providing more opportunity for methanogenic archaea than in other non‐ruminant mammals to produce significant amounts of methane.     

悉生条件下3株嗜盐古菌对卤虫生长及其抗弧菌感染的影响

嗜盐古菌存在于高盐水体中,是高盐环境微生物种群的重要组成部分。本研究利用从日晒盐场结晶池卤水中分离得到的3株红色嗜盐古菌,分别命名为Haloarcula sp.HG-1、Haloferax sp.KN-4和Halorubrum sp.IT-5。采用悉生实验系统,探讨不同盐度(30、100和150)条件下3株古菌能否作为卤虫(Artemia)的唯一饵料,并比较其作为饵料对卤虫存活、生长和抗鳗弧菌(Vibrio anguillarum)感染能力的影响。结果表明,在盐度30的悉生系统中,投喂3株古菌组卤虫存活率和体长均保持较高水平;鳗弧菌攻毒条件下,卤虫存活率有所下降,其中投喂Haloferax sp.KN-4菌株组卤虫抗鳗弧菌感染能力最强。正常和攻毒条件下,在盐度100和盐度150悉生系统中分别投喂3株古菌,卤虫均保持较高存活率和体长,其中投喂Haloarcula sp.HG-1菌株组卤虫具有最高存活率和最长体长,但攻毒组卤虫存活率和生长普遍优于未攻毒组,这可能与高盐条件下V.anguillarum毒力下降有关。本研究利用"卤虫-古菌"悉生实验系统,证实了3株嗜盐古菌均可作为卤虫的唯一饵料,为卤虫提供营养;利用"卤虫-古菌-致病菌"攻毒实验系统,证实3株嗜盐古菌均可以增强卤虫抗鳗弧菌感染能力。本研究结果为研究古菌在高盐环境食物链中的作用提供了基础数据。     

秸秆直接还田与炭化还田对潮土硝化微生物的影响

为比较秸秆直接还田和炭化还田对黄淮海平原小麦/玉米轮作体系典型潮土硝化作用及硝化微生物群落的影响,设置4个处理:全量小麦秸秆还田(S)、全量秸秆炭化还田(B)、半量秸秆半量生物质炭还田(SB)和不进行秸秆或生物质炭还田的对照(CK),连续进行3 a田间试验。对小麦、玉米两个生长季土壤理化性质进行分析,用末端限制性片段长度多态性(Terminal-restriction length polymorphism,T-RFLP)技术和克隆文库技术对氨氧化古菌(Ammonia-oxidizing archaea,AOA)和氨氧化细菌(Ammonia-oxidizing bacteria,AOB)群落结构和多样性进行分析。结果表明,在小麦季,与S处理相比,B处理显著降低了土壤容重,提高了土壤pH、有机碳(SOC)和速效钾(AK)含量(P0.05),但并未显著影响土壤水分、铵态氮(NH_4~+-N)和硝态氮(NO_3~--N)含量;B和SB处理的硝化潜势(Potential nitrification rate,PNR)分别为0.58、0.49μg·h~(-1)·g~(-1)(以NO_2~-计,下同),显著高于CK,与S处理(0.40μg·h~(-1)·g~(-1))差异不显著。玉米季,B处理显著提高了土壤水分、SOC和AK(P0.05),各处理玉米季的PNR整体低于小麦季,B处理最高(0.27μg·h~(-1)·g~(-1)),显著高于CK和S处理(P0.05)。小麦季PNR分别与AK、NH_4~+浓度和土壤容重显著相关(P0.05),与AOA和AOB群落组成均无显著关系;玉米季PNR仅与理化因子SOC显著相关,但该季节PNR与AOB群落结构显著相关。冗余分析(RDA)表明,土壤SOC、容重、pH和AK是显著影响硝化微生物群落结构的主要因子,对AOA和AOB群落结构总变异的解释量分别为76.4%和75.5%。系统发育树分析表明,AOA大部分属于土壤古菌Group1.1b,AOB多属于亚硝化螺菌Nitrosospira簇3。综上,与秸秆直接还田相比,炭化还田提高土壤硝化活性,改善部分土壤理化性质,引起土壤硝化微生物群落结构变化。     

黄顶菊入侵对土壤氨氧化古菌群落多样性的影响

为了明确入侵植物黄顶菊对土壤氮循环关键过程硝化作用的影响机制,本研究通过对其入侵地和未入侵地、根围土和根际土氨氧化古菌(ammonia-oxidizing archaea,AOA)的群落多样性分析,探讨了AOA对黄顶菊入侵的响应规律。结果表明:黄顶菊入侵增加了入侵地根围土AOA的多样性,AOA的Shannon指数表现为:入侵地根围土入侵地根际土未入侵地土壤,且差异显著。铵态氮含量与p H的变化都会影响土壤AOA的群落结构。系统发育树分析表明,土壤AOA主要隶属于氨氧化古菌的Nitrososphaera cluster。黄顶菊入侵导致的AOA的多样性水平的提升主要是由于入侵地氨氧化菌群种类增加所致。     

模拟条件下土壤硝化作用及硝化微生物对不同水分梯度的响应

以江苏滨海县一植稻土壤为研究对象,在微宇宙培养条件下设置了不同水分处理(最大持水量的30%、60%、90%和淹水2 cm深),研究了硝化作用及硝化微生物对水分变化的响应特征。结果表明:淹水处理显著降低了土壤的氧化还原电位(Eh),但所有处理土壤Eh变化范围为330~500 m V,土壤整体处于氧化态。在每7天向土壤加入10 mg kg-1NH+4-N的连续培养过程中,各个水分处理均观察到明显的NH+4-N降低和NO-3-N累积的现象,60%WHC处理下土壤硝态氮累积最显著和迅速,90%WHC处理次之,随培养时间延长,30%WHC和淹水处理也观察到明显的硝化作用。淹水处理中氨氧化细菌(Ammonia-oxidizing bacteria,AOB)的数量显著高于非淹水处理,且淹水处理中AOB在DGGE图谱上的条带更加清晰明亮,而氨氧化古菌(Ammonia-oxidizing archaea,AOA)的群落组成和数量在不同水分处理间无明显变化。表明该土壤中AOB对水分条件变化响应灵敏,是该土壤的硝化作用、尤其是淹水条件下硝化作用发生的主要原因。