張小梅 咸洪泉 李雅華 陳清華
摘要:為深入研究堆肥過程中主要微生物的功能,以番茄秸稈為原料,對(duì)其反應(yīng)器堆肥過程進(jìn)行研究。通過反應(yīng)器自動(dòng)溫度傳感器記錄堆肥溫度,利用分光光度法測(cè)定堆肥過程中植物生物質(zhì)降解相關(guān)酶活力,并對(duì)酶活力最高時(shí)期的典型代表性樣品進(jìn)行高通量Orbitrap質(zhì)譜鑒定。結(jié)果表明,反應(yīng)器堆肥共持續(xù)35天,其中50℃以上的高溫期10天以上,發(fā)酵至第7天時(shí),內(nèi)切纖維素酶和木聚糖酶活力最高。對(duì)該時(shí)期樣品進(jìn)行質(zhì)譜鑒定發(fā)現(xiàn),Thermobifida是主要的功能細(xì)菌,且是唯一的纖維素降解者,產(chǎn)生糖苷水解酶(GH)5家族和GH9家族的5種內(nèi)切纖維素酶和3種GH48家族的外切纖維素酶。Planifilum產(chǎn)生GH3家族的β-葡萄糖苷酶,與Thermobifida一起將纖維素降解成葡萄糖。Thermobifida、Planifilum、Saccharomonospora、Aspergillus和Thermomyces共同參與木聚糖的降解,前三者為功能細(xì)菌,主要分泌GH10家族的木聚糖酶,后二者為功能真菌,主要分泌GH11家族的木聚糖酶。
關(guān)鍵詞:堆肥;糖苷水解酶;功能微生物;高通量質(zhì)譜
中圖分類號(hào):S141.4文獻(xiàn)標(biāo)識(shí)號(hào):A文章編號(hào):1001-4942(2020)04-0073-06
Abstract To further identify functions of key microorganisms in composting, the reactor composting process using tomato straw as raw material was studied. The compost temperature was auto-monitored by the temperature sensor, and the spectrophotometric method was applied in determining activities of the lignocellulosic enzymes. The sample with the highest activity was selected as the typical representative for further identification by high-throughput Orbitrap-LC-MS/MS. The results showed that, the thermophilic phase with temperature >50℃?lasted for more than 10 days during the whole period of 35 days, and the activities of both endoglucanase and xylanase peaked on the 7th day. The Orbitrap analysis revealed that Thermobifida was the only cellulose degrader secreting five endoglucanases including members of glycoside hydrolase (GH) family 5 and GH family 9, and three cellobiohydrolases from GH48. Planifilum cooperated with Thermobifida in cellulose degrading to glucose by secreting β-glucosidase from GH family 3. Thermobifida, Planifilum, Saccharomonospora, Aspergillus and Thermomyces participated in xylan degrading. Xylanases produced by the former three bacteria were members of GH10, and those secreted by the latter two fungi were from GH11.
Keywords Composting; Glycoside hydrolase; Functional microorganism; High-throughput mass spectrometer
堆肥是一種處理固體有機(jī)廢物的有效途徑,是系列微生物相互協(xié)同、共同作用的結(jié)果[1]。堆肥工藝的優(yōu)化、宏觀參數(shù)的調(diào)控實(shí)際上都是為更好地促進(jìn)好氧微生物的活性[2]。
酶是堆肥發(fā)酵過程中微生物參與代謝活動(dòng)的直接體現(xiàn)。酶活性越高,對(duì)應(yīng)微生物參與的代謝活動(dòng)越旺盛。檢測(cè)酶活力的方法,常見的是分光光度法,它通過檢測(cè)一定時(shí)間內(nèi)底物的減少量或產(chǎn)物的增加量來反映酶活力的高低[3],該方法簡(jiǎn)單快速、實(shí)用性強(qiáng)。然而,在由多種微生物組成的復(fù)雜生境(如堆肥、土壤)系統(tǒng)中,往往有多種微生物參與同一代謝過程。如在堆肥生態(tài)系統(tǒng)中,參與纖維素降解的微生物既有細(xì)菌,如Thermobifida,又有真菌,如Aspergillus[4,5]。因此,對(duì)于復(fù)雜生境而言,僅通過酶活力的測(cè)定無法真實(shí)表征微生物的代謝活動(dòng)。
宏蛋白質(zhì)組(metaproteomics)是指特定時(shí)刻下,環(huán)境微生物所表達(dá)的所有蛋白[6]。因其直接以參與各種代謝活動(dòng)的功能實(shí)體(functional entities)——蛋白質(zhì)為研究對(duì)象,同時(shí)無需蛋白分離、可直接對(duì)混合樣品中的全部組分進(jìn)行分析,故可原位檢測(cè)微生物的功能、更加有效地探明微生物的具體代謝過程[7]。宏蛋白質(zhì)組學(xué)分析一般依賴質(zhì)譜儀,尤其是近年來,高性能、高靈敏度新質(zhì)譜儀(如LTQ-Orbitrap)的推陳出新為新一代宏蛋白質(zhì)組學(xué)技術(shù)在復(fù)雜生境研究中的廣泛應(yīng)用開辟了新的視野[4,5,8]。
基于此,本研究以番茄秸稈反應(yīng)器堆肥為研究對(duì)象,通過基礎(chǔ)理化性質(zhì)和木質(zhì)纖維素降解酶活力的監(jiān)測(cè),確定腐解效率最高時(shí)期的典型代表性樣品,并對(duì)其進(jìn)行質(zhì)譜鑒定以檢測(cè)胞外蛋白組成及豐度。這對(duì)進(jìn)一步明確番茄秸稈堆肥過程中主要微生物的代謝功能具有重要意義。
1 材料與方法
1.1 儀器與試劑
儀器:LTQ-Orbitrap-LC-MS/MS(將Shimadzu高效液相色譜儀Prominence nano液相色譜系統(tǒng)和ThermoFisher線性離子阱質(zhì)譜儀LTQ-Orbitrap Velos Pro ETD相偶聯(lián))、高速冷凍離心機(jī)、分光光度計(jì)、電熱恒溫水浴鍋、冷凍干燥儀。
藥品和試劑:二硫蘇糖醇(DTT)、乙二胺四乙酸(EDTA)、三氯乙酸(TCA)、三氟乙酸(TFA)、鹽酸胍、碘乙酰胺(IA)、乙腈、Trypsin、NH4HCO3、冰醋酸均購(gòu)自Sigma。
變性緩沖液:0.5 mol/L Tris-HCl,2.75 mmol/L?EDTA,6 mol/L鹽酸胍溶于10 mL超純水中,調(diào)pH至8.1±0.1。
1.2 數(shù)據(jù)庫(kù)與軟件
Uniprot數(shù)據(jù)庫(kù)(https://www.uniprot.org/),Proteome Discovered Software 1.4 (Thermo Fisher Scientific)質(zhì)譜結(jié)果比對(duì)軟件。
1.3 反應(yīng)器堆肥試驗(yàn)設(shè)置
從校試驗(yàn)田收集新鮮番茄秸稈,充分晾曬切成2~3 cm小段后,測(cè)定其pH值、有機(jī)質(zhì)(OM)、總氮(TN)、碳氮比(C/N);之后,據(jù)測(cè)定結(jié)果以玉米秸稈為調(diào)理劑,調(diào)節(jié)物料的C/N比值在20~30之間、含水率(MC)至50%~60%。準(zhǔn)備好的物料裝入直徑為200 mm、高500 mm、有效工作體積為15.7 L的堆肥反應(yīng)器內(nèi)(反應(yīng)器由一臺(tái)200 W氣泵供氣,帶溫度和氧傳感器,可自動(dòng)測(cè)量堆肥溫度、進(jìn)氣和排氣中O2濃度)。蓋好上蓋,啟動(dòng)氣泵通氣,控制通風(fēng)量為0.2 m3/(min·m3物料)左右。
觀察并記錄堆溫的變化,當(dāng)堆溫由環(huán)境溫度上升到60~70℃之后下降至接近環(huán)境溫度不再變化時(shí),終止通氣。將物料取出,進(jìn)行第一次翻堆,材料充分翻動(dòng)、混合后再放回反應(yīng)器中,蓋好上蓋,重新啟動(dòng)氣泵通氣。
1.4 取樣
堆肥試驗(yàn)共持續(xù)35天。從堆肥發(fā)酵之初(0天),每隔一周采用“五點(diǎn)取樣法”采集堆肥樣品,混合均勻后作為該時(shí)期樣品。
1.5 酶活測(cè)定
取各時(shí)期樣品100 g,加入400 mL蒸餾水,4℃靜置24 h 后紗布過濾,
8 000 r/min離心10 min,去沉淀留上清即得粗酶液。采用二硝基水楊酸(DNS)法測(cè)定各時(shí)期粗酶液的內(nèi)切纖維素酶和木聚糖酶活性[9]。所有試驗(yàn)重復(fù)3次。以吸光度的平均值±標(biāo)準(zhǔn)差表征酶活性的高低。
1.6 質(zhì)譜
1.6.1 樣品預(yù)處理及上機(jī) 以1.5中測(cè)得的酶活力最高時(shí)期的典型代表性堆肥樣品進(jìn)行Orbitrap質(zhì)譜鑒定。樣品預(yù)處理流程詳見圖1[10]。肽段噴入質(zhì)譜儀的電壓為2 000 V,轉(zhuǎn)移毛細(xì)管的溫度為275℃,質(zhì)荷比(m/z)和分辨率分別設(shè)為400和60 000,得到全掃描質(zhì)譜。
1.6.2 生物信息學(xué)分析 查閱相關(guān)文獻(xiàn),初步明確已有文獻(xiàn)報(bào)道的番茄秸稈堆肥過程中主要的細(xì)菌屬和真菌屬。從Uniprot數(shù)據(jù)庫(kù)(http://www.uniprot.org)中下載優(yōu)勢(shì)屬的fasta格式蛋白質(zhì)組數(shù)據(jù)文件,形成質(zhì)譜數(shù)據(jù)比對(duì)的參考數(shù)據(jù)庫(kù)。用Thermo Proteome Discovered Software 1.4對(duì)質(zhì)譜數(shù)據(jù)進(jìn)行比對(duì)分析。錯(cuò)配閾值設(shè)為0.05。每個(gè)蛋白的Accession號(hào)唯一,Description描述了該蛋白的功能及其來源微生物,通過peptide spectrum matching (PSM)參數(shù)定量表征對(duì)應(yīng)蛋白的豐度。
2 結(jié)果與分析
2.1 堆肥溫度的變化
堆肥反應(yīng)器溫度變化如圖2所示??梢钥闯?,堆溫在開始的前7天內(nèi)不斷上升,至第5天時(shí)溫度已高于50℃,進(jìn)入高溫期,至第7天時(shí)溫度最高,為62℃,之后開始下降;50℃以上的高溫期持續(xù)了10天以上,之后堆溫繼續(xù)降低,在第25天時(shí)下降到與室溫基本相當(dāng)。物料重新混勻后再次發(fā)酵,溫度又有所上升,但最高溫仍低于50℃,然后又很快下降到與室溫相當(dāng),發(fā)酵結(jié)束。
2.2 酶活力變化
檢測(cè)了堆肥發(fā)酵過程中與秸稈類植物生物質(zhì)降解最相關(guān)的兩種酶——內(nèi)切纖維素酶和木聚糖酶活力的變化,結(jié)果如圖3所示??梢钥闯?,發(fā)酵開始的第一周內(nèi)這兩種酶活力不斷上升,至第7天時(shí)酶活力最高,OD550分別為0.52±0.03和0.73±0.04,之后酶活力開始緩慢下降;內(nèi)切纖維素酶在二次發(fā)酵過程中,活力又小幅上升,但OD550也僅為最高酶活力時(shí)的60%左右。由于第7天時(shí)兩種與秸稈類植物生物質(zhì)降解最相關(guān)的酶活力最高,一定程度上表明該時(shí)期參與秸稈降解的微生物代謝活性最為旺盛,故選該時(shí)期樣品作為典型代表性樣品進(jìn)行質(zhì)譜鑒定,以探明秸稈降解過程中關(guān)鍵微生物的功能。
2.3 主要微生物和酶的鑒定
通過查閱已發(fā)表的番茄秸稈堆肥相關(guān)文獻(xiàn)[11-14],初步明確黃桿菌屬(Flavobacterium)、副球菌屬(Paracoccus)、擬諾卡氏菌屬(Nocardiopsis)、糖霉菌屬(Glycomyces)、馬杜拉放線菌屬(Actinomadura)、芽孢桿菌屬(Bacillus)、喜熱裂孢菌屬(Thermobifida)、糖單孢菌屬(Saccharomonospora)和清野氏菌屬(Planifilum)共計(jì)11個(gè)細(xì)菌屬,曲霉屬(Aspergillus)、嗜熱絲孢菌屬(Thermomyces)、支頂孢屬(Acremonium)和毛殼屬(Chaetomium)共計(jì)4個(gè)真菌屬,可能是堆肥發(fā)酵過程中的關(guān)鍵微生物。遂從Uniprot數(shù)據(jù)庫(kù)中下載以上微生物屬的蛋白質(zhì)組序列信息作為質(zhì)譜數(shù)據(jù)比對(duì)的參考數(shù)據(jù)庫(kù)。比對(duì)后的質(zhì)譜結(jié)果如見表1。
[6] Maron P, Ranjard L, Mougel C, et al. Metaproteomics: a new approach for studying functional microbial ecology [J]. Microbial Ecology, 2007, 53(3): 486-493.
[7] Kucharova V, Wiker H. Proteogenomics in microbiology: taking the right turn at the junction of genomics and proteomics [J]. Proteomics, 2014, 14: 2360-2675.
[8] Pore S, Engineer A, Dagar S, et al. Meta-omics based analyses of microbiome involved in biomethanation of rice straw in a thermophilic anaerobic bioreactor under optimized conditions [J]. Bioresource Technology, 2019, 279: 25-30.
[9] 張小梅.糖苷水解酶性質(zhì)高通量分析平臺(tái)的建立及GH12家族酶組分多功能活性架構(gòu)的機(jī)理研究[D].濟(jì)南:山東大學(xué),2013.
[10]Godoy L, Olsen J, Nielsen M, et al. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast [J]. Nature, 2008,455: 1251-1254.
[11]張津,劉微,李博文.番茄秸稈和雞糞堆腐中微生物菌劑的篩選[J].河北農(nóng)業(yè)大學(xué)學(xué)報(bào),2013,36(5):76-81.
[12]劉微,張津,李博文, 等.不同微生物菌劑對(duì)番茄秸稈好氧堆肥中氮磷鉀元素的轉(zhuǎn)化規(guī)律的影響[J].中國(guó)土壤與肥料,2014(3):88-92.
[13]Liu W, Wang S, Zhang J, et al. Biochar influences the microbial community structure during tomato stalk composting with chicken manure [J]. Bioresource Technology, 2014, 154:148-154.
[14]Alkoaik F, Ghaly A E. Influence of dairy manure addition on the biological and thermal kinetics of composting of greenhouse tomato plant residues [J]. Waste Management, 2006, 26(8):902-913.
[15]Biely P. Microbial xylanolytic systems [J]. Trends in Biotechnology, 1985, 3(11): 286-290.
[16]Gong W L, Zhang H Q, Tian L, et al. Determination of the modes of action and synergies of xylanases by analysis of xylooligosaccharide profiles over time using fluorescence-assisted carbohydrate electrophoresis [J]. Electrophoresis,2016,37(12):183-189.
[17]Dodd D, Cann I K O. Enzymatic deconstruction of xylan for biofuel production[J]. GCB Bioenergy, 2009, 1:2-17.
[18]Kolenova K,Vrsanska M,Biely P.Mode of action of endo-β-1,[JP]4-xylanases of families 10 and 11 on acidic xylooligosaccharides [J]. Journal of Biotechnology, 2006, 121(3):338-345.
[19]Bogusawa K, Dagmara K, Stanisaw B. Efficient expression and secretion of two co-produced xylanases from Aspergillus niger in Pichia pastoris directed by their native signal peptides and the Saccharomyces cerevisiae alpha-mating factor[J]. Enzyme & Microbial Technology, 2006, 39(4):683-689.
[20]Meng D D, Ying Y, Chen X H, et al. Distinct roles for carbohydrate-binding modules of glycoside hydrolase 10 (gh10) and gh11 xylanases from Caldicellulosiruptor sp. strain F32 in thermostability and catalytic efficiency [J]. Applied and Environmental Microbiology, 2015, 81(6):2006-2014.