Accelerating carbon conversion in garden waste composting with food waste-expanding microbial inoculants
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摘要: 高木质纤维素含量制约了园林废弃物的堆肥化应用, 添加外源菌剂是加快木质纤维素降解的有效手段。为降低菌剂生产成本并提高接种效率, 本研究利用食品残渣(苹果渣、豆渣)代替常规碳氮源(葡萄糖、蛋白胨)进行木质纤维素降解菌的扩繁, 扩繁产品活菌数高达3.7×1010 cfu∙mL−1, 相较工业培养基增加46.2%。进一步探讨了不同降解菌接种量(0、2%、4%、8%, 干重计)对园林废弃物堆肥过程碳素转化的影响。结果表明, 接种处理显著增加了木质纤维素的降解(P<0.05), 2%、4%和8%接种处理(2%IM、4%IM、8%IM)的总木质纤维素降解率较CK分别提高6.3%、9.2%和23.0%; 其中8%IM处理加速了碳素的完全矿化, 导致腐殖质前体物(多酚、还原糖)被完全降解生成CO2, 抑制了腐殖化的进行; 而4%IM处理在加快木质纤维素降解的同时促进了腐殖酸(HA)的合成, 其最终HA含量达91.3 g∙kg−1, 较CK、2%IM和8%IM处理分别提高24.9%、10.7%和35.8%。因此, 以食品残渣为培养基质可完全实现木质纤维素降解菌的生长扩繁, 同时, 4%接种量更有利于园林废弃物堆肥腐殖化的进行和碳素的保存, 本研究为多源废弃物高效协同处理提供了理论依据。Abstract: The expansion of urbanization has resulted in the generation of a large amount of garden waste (40 million tons per year in China), while traditional treatment methods (incineration and landfill) tend to cause serious environmental pollution and waste of resources. Composting is an effective way to realize resource utilization of garden waste. However, the high lignocellulose content of garden waste limits its resource utilization. Accelerating the degradation of lignocellulose in the composting process is of great significance for achieving effective resource utilization of garden waste. Inoculation with exogenous microorganisms is considered an environmentally friendly and cost-effective method to accelerate lignocellulose degradation, which would further reduce the cost of inoculum production and improve inoculation efficiency. In this study, food residues (apple pomace and bean dregs) were used instead of conventional carbon and nitrogen sources (glucose and peptone) to propagate lignocellulose-degrading fungi. The number of viable fungi in the multiplication product reached 3.7×1010 cfu∙mL−1, which increased by 46.2% compared with the traditional industrial medium. The effects of different inoculum amounts (0, 2%, 4%, and 8%, dry weight) on carbon conversion during garden waste composting were also discussed. The inoculation treatments significantly increased lignocellulose degradation (P<0.05), according to the results. The total lignocellulose degradation rates of the 2%, 4%, and 8% inoculation treatments (2%IM, 4%IM, and 8%IM) increased by 6.3%, 9.2%, and 23.0%, respectively, compared with CK. Dynamic changes in humus precursors (reducing sugars and polyphenols) and humus components were further analyzed. The 8%IM treatment accelerated the complete mineralization of carbon, resulting in the complete degradation of the humus precursors (polyphenols and reducing sugars) into CO2, which inhibited humification. Compared with CK, 2%IM, and 4%IM, the cumulative CO2 emissions of 8%IM increased by 21.9%, 22.3%, and 26.0%, respectively. The 4%IM treatment accelerated lignocellulose degradation while promoting the synthesis of humic acid (HA). The final HA content reached 91.3 g∙kg−1, which was 24.9%, 10.7%, and 35.8% higher than that of CK, 2%IM, and 8%IM treatments, respectively. These results indicate that appropriate inoculation is beneficial to the directional transformation of lignocellulose to humic acid, whereas excessive inoculation would lead to an excessive loss of organic matter due to the high metabolic activity of microorganisms; and the degradation efficiency of lignocellulose is lower when inoculated with a small amount, which was further confirmed by the partial least squares path analysis model in this study. The conversion of lignocellulose to dissolved organic carbon increased with increasing inoculation amount (correlation coefficients of CK, 2%IM, 4%IM, and 8%IM were 0.59, 0.70, 0.75, and 0.85, respectively), while the correlation coefficient of 4%IM from DOC to HA was −0.85, which was higher than 2%IM (−0.76) and 8%IM (−0.34). Therefore, the growth and propagation of lignocellulose-degrading fungi can be completely realized by using food residues as a culture substrate. A 4% inoculation amount was more conducive to the humification of garden waste compost and the preservation of carbon. This study provides a reference for the garden waste composting inoculation process and a theoretical basis for multi-source waste-efficient collaborative treatment.
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Keywords:
- Composting /
- Garden waste /
- Carbon transformation /
- Food waste /
- Lignocellulose degradation /
- Humification
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城镇化的扩张造成了大量园林废弃物(0.4亿t)的产生, 而传统处理方式(焚烧、填埋)极易造成严重的环境污染和资源浪费[1-2]。众所周知, 堆肥是一种快速实现有机物稳定化和无害化的处理方式, 同时兼顾环境和经济效益[3]。园林废弃物具有孔隙度大、含水率低、C/N高的特点, 难以进行单独堆肥, 这些缺点均可通过与其他废弃物(餐厨垃圾、畜禽粪便)进行联合堆肥的方式予以解决[4-5]。然而园林废弃物中木质纤维素含量较高, 不利于微生物分解利用, 影响堆肥发酵速率, 成为制约园林废弃物堆肥化利用的限制因素[6], 因此, 加快堆肥过程中木质纤维素的降解对实现园林废弃物高效资源化利用具有重要意义。
目前, 研究者们已开发了多种加速堆肥过程中木质纤维素降解的手段, 如Fenton法[7]、水热预处理[8]、接种外源微生物[9]等, 其中接种外源微生物被认为是最为环境友好且经济有效的方式[10]。微生物类型包含细菌、真菌、放线菌的一种或多种[11], 目前微生物菌剂的研发多侧重于开发新型菌种, 而针对微生物扩繁活化过程的研究却相对较少。菌剂扩繁培养时通常以葡萄糖、蛋白胨等物质作为碳氮源, 在增加扩繁成本的同时也间接提高了废弃物堆肥处理成本, 因此, 实现微生物菌剂的低成本扩繁至关重要。食品工业的发展造成了大量食品残渣的产生, 如果渣、豆渣等, 食品残渣中含有丰富的糖分和蛋白质, 可替代葡萄糖、蛋白胨成为良好的微生物培养底物, 降低菌剂生产成本的同时实现食品残渣的处理, 然而其扩繁效率尚未可知, 有待进一步研究。
堆肥过程中碳素的转化主要包括大分子有机质的水解、CO2释放和腐殖化以及厌氧产CH4 4个过程。其中木质纤维素等大分子物质在胞外酶的作用下被逐渐分解为小分子物质, 如还原糖、多酚等[12], 小分子物质一方面被微生物完全矿化生成CO2并排放到空气中, 另一方面可通过特定途径聚合形成大分子腐殖质(HS)[13], HS对提高堆肥产品肥力和稳定性具有重要作用[14], 而在先进的堆肥方式和供气策略下, CH4的排放可被有效避免[15]。目前, 已有研究表明, 外源微生物的接种量会影响微生物菌剂功能的有效发挥[16], 而接种量与园林废弃物堆肥过程中木质纤维素的降解、CO2释放以及HS的形成之间的关系尚不清楚。因此, 本研究以食品残渣(苹果渣、豆渣)为基质进行木质纤维素降解菌的扩繁, 并探讨了不同接种量对园林废弃物堆肥过程中碳素转化的影响, 为实现微生物低成本扩繁、园林废弃物高效堆肥以及多源废弃物的协同处理提供理论依据。
1. 材料与方法
1.1 试验材料
堆肥试验原料为园林废弃物与餐厨垃圾, 园林废弃物(修剪后的树枝和落叶)取自中国科学院栾城农业生态系统试验站, 粉碎3~5 cm后备用; 餐厨垃圾取自于石家庄栾城县城某饭店, 苹果渣通过新鲜苹果榨汁后获得, 豆渣取自石家庄市栾城镇小任家庄村豆腐坊。原材料的基本理化性质如表1所示。
表 1 试验材料的理化性状Table 1. Physical and chemical properties of the experimental materials试验材料
Material含水率
Moisture
content (%)有机质a
Organic
matter (%)总氮a
Total
nitrogen (%)碳氮比a
C/NpH 电导率
Electrical
conductivity (mS·cm−1)总糖a
Total sugar
content (%)园林废弃物
Garden waste11.00±0.34 84.74±0.56 1.49±0.00 34.14±0.20 7.32±0.02 1.55±0.17 — 餐厨垃圾
Food waste78.55±2.23 91.74±0.11 2.89±0.02 14.61±0.32 4.58±0.07 3.30±0.20 — 苹果渣
Apple pomace82.81±0.58 98.04±0.14 0.81±0.02 69.87±1.82 5.67±0.09 — 20.75±0.58 豆渣
Bean dregs79.95±4.14 95.73±0.07 2.64±0.01 21.02±0.09 7.07±0.04 — 37.29±1.29 “a”: 基于物料干重; “—”: 表示未检测。“a”: based on dry weight of material; “—”: not measured. 1.2 菌剂制备
微生物菌种: 木质纤维素降解菌株选用2株耐高温真菌, 均从高温期堆肥物料中筛选获得, 分别为草酸青霉(Penicillium oxalicum)和米曲霉(Aspergillus oryzae)。
菌剂种子液制备: 选用马丁氏培养基, 培养基配方为葡萄糖10 g, 蛋白胨5 g, 酵母浸出粉2 g, 磷酸氢二钾1 g, 硫酸镁0.5 g, 蒸馏水1 L; 经121 ℃高压灭菌21 min后备用, 将保存的菌株接种于培养基中, 45 ℃、100 rpm条件下摇床培养72 h后备用。
发酵培养基: 培养基Ⅰ选用常规工业碳氮源为底物, 配方为葡萄糖20 g, 蛋白胨5 g, 蒸馏水1 L; 培养基Ⅱ选用食品残渣(苹果渣、豆渣)作为碳氮组分, 以食品残渣中总糖替代葡萄糖, 食品残渣中的总氮替代蛋白胨, 配方为苹果渣300 g, 豆渣100 g (鲜重计), 碳酸钙3 g, 蒸馏水1 L, 其中添加CaCO3是为了避免食品残渣在发酵过程中产生过量有机酸, 导致培养基pH下降, 从而影响微生物的生长, 培养基经121℃高温灭菌21 min后冷却使用。
培养过程: 将种子液按3%的比例分别接种于不同发酵培养基中, 每种培养基设置3组重复, 将培养基置于45 ℃, 100 rpm条件下摇床培养72 h后; 每12 h在无菌条件下取样, 取样时采用灭菌的移液器吸取10 mL样品液置于灭菌的离心管中, 后续进行活菌数、木聚糖酶、纤维素酶、木质素过氧化物酶、锰过氧化物酶、漆酶的测定。
1.3 堆肥试验
堆肥试验采用50 L不锈钢堆肥反应器装置进行。反应器系统详细描述参考文献[17]。堆肥物料以园林废弃物为主料, 餐厨垃圾为辅料, 园林废弃物与餐厨垃圾按4∶1比例充分混合(干重比), 堆肥试验共设置4个处理: 不添加菌剂的对照(CK), 添加2% (2%IM)、4% (4%IM)、8% (8%IM)的菌剂处理, 添加比例为每千克物料(干重计)所添加菌液的质量(kg, 鲜重计), 添加菌剂为发酵培养基Ⅱ扩繁培养后所获得的菌液。堆肥初始物料含水率调节至60%左右, 通气速率设置为0.2 L∙min−1∙kg−1 (干物质重), 进行35 d的好氧堆肥试验, 并在堆肥第0 d、4 d、7 d、14 d、21 d、28 d、35 d进行翻堆取样, 翻堆时将堆肥物料进行人工混合, 并采用多点取样法进行取样, 每次采集样品量为200 g, 采集的样品分为两份, 一份用于基本理化指标的测定, 另一份进行冷冻保存。
1.4 样品分析
活菌数的测定采用平板计数法[18]; 木聚糖酶、纤维素酶活的测定参考文献[19], 酶活性单位(U)定义为1 min分解底物生成1 μg还原糖所用的酶量; 木质素降解相关酶活力测定(漆酶、锰过氧化物酶和木素过氧化物酶)活力测定参照田林双[20]的方法, 酶活力单位(U)为1 min分解1 μmol底物所使用的酶量。
堆肥温度通过温度传感器实时检测, 温度传感器位于反应器中心, 每隔1 h自动检测堆体温度; 含水率通过105 ℃烘干法测得; 采用流动分析仪(Vario TOC select, Elementar, Germany)测定堆肥浸提液中的可溶性有机碳(DOC)含量; CO2于每天上午10:00在堆肥反应器出气口处利用气袋进行收集, 并通过气相色谱测定其浓度[17]。
部分堆肥样品经风干后用于理化指标的测定, 其中有机质(OM)的测定采用马弗炉550 ℃灼烧法, 灼烧后剩余部分为灰分含量; 全氮(TN)含量采用凯氏定氮法测定; 还原糖采用DNS比色法测定[21]; 多酚测定方法参考文献[22]。
木质纤维素含量依据范式洗涤法原理[23], 采用全自动纤维素分析仪测定; 根据灰分守恒原理计算木质纤维素的降解率, 降解公式如下[24]:
$$ l=1-\left(X_1 \times C_2\right) /\left(X_2 \times C_1\right) $$ (1) 式中: l为木质纤维素降解率(%), X1、 X2为初始和最终灰分含量, C1、C2为初始和最终木质纤维素含量。
腐殖质(HS)含量及组分[腐殖酸(HA)和富里酸(FA)]测定采用絮凝容量法[23], 腐殖化指数(HI)、聚合度(DP)、腐殖酸占比(PHA)计算公式如式3、式4和式5所示[12]:
$$ C_{{\rm{HS}}}=C_{{\rm{HA}}}+C_{{\rm{FA}}} $$ (2) $$\mathrm{HI}( {\text{%}} )=C_{\mathrm{HA}} / \mathrm{TOC} \times 100$$ (3) $$ \mathrm{DP}=C_\mathrm{HA}/C_\mathrm{FA} $$ (4) $$ \mathrm{PHA}=C_\mathrm{HA}/C_\mathrm{HS} $$ (5) 式中: CHS为腐殖质碳含量, CHA为腐殖酸碳含量, CFA为富里酸碳含量, TOC为总有机碳含量。
1.5 数据处理
采用Excel 2010进行试验数据处理和图表绘制, 采用SPSS 21.0统计软件进行显著性和相关性分析, 使用IBM SPSS Amos 26软件进行偏最小二乘法路径模型分析。
2. 结果与分析
2.1 不同培养基对菌种扩繁及酶活力的影响
微生物菌种经过72 h的好氧扩繁后, 有效活菌数均达到1010 cfu∙mL−1以上(表2), 符合GB 20287—2006《农用微生物菌剂》的标准(>109 cfu∙mL−1), 其中培养基Ⅱ的最终活菌数相较于培养基Ⅰ显著增加46.2% (P<0.05), 表明食品残渣(苹果渣、豆渣)可有效代替常规碳、氮源, 成为良好的微生物培养底物; 同时食品残渣中含有丰富的维生素和矿物质[25-26], 更有利于微生物的生长代谢。
表 2 食品残渣(苹果渣、豆渣)代替常规碳、氮源(培养基 Ⅱ)对培养72 h后菌液活菌数与酶活性的影响Table 2. Effect of food residues (apple pomace, soybean dregs) instead of conventional carbon and nitrogen sources (Medium Ⅱ) on the viable count and enzymatic activity of the broth after 72 h incubation处理
Treatment活菌数
Viable count
(×010 cfu∙mL−1)木聚糖酶
Xylanase
(U∙mL−1)纤维素酶
Cellulase
(U∙mL−1)漆酶
Laccase
(U∙mL−1)锰过氧化物酶
Manganese peroxidase
(U∙mL−1)木质素过氧化物酶
Lignin peroxidase
(U∙mL−1)培养基Ⅰ Medium Ⅰ 2.53±0.41a 337.40±108.81a 73.82±9.55a 3.71±0.69a 11.26±3.97a 2.98±1.67a 培养基Ⅱ Medium Ⅱ 3.70±0.37b 597.40±171.21b 99.47±5.99b 4.91±2.22a 22.38±2.46b 12.39±1.67b 不同小写字母表示不同处理间存在显著性差异(P<0.05)。Different lowercase letters indicate significant differences between two treatments (P<0.05). 木聚糖酶、纤维素酶、木质素降解酶(漆酶、锰过氧化物酶、木质素过氧化物酶)分别与半纤维素、纤维素、木质素的降解有关[27]。如表2所示, 培养基Ⅱ的木质纤维素降解相关酶活均高于培养基Ⅰ, 表明以食品残渣(苹果渣、豆渣)为底物更有利于各组分酶的分泌表达; 苹果渣、豆渣中均含有一定量的粗纤维成分[25-26], 可作为微生物产生木质纤维素相关酶的诱导底物[28], 这在前人研究中也有所报道: 如于俊杰等[29]以玉米秸秆为底物刺激木聚糖酶的产生; 王晓芳等[28]以微晶纤维素为底物刺激纤维素酶的产生; 毕杨等[30]以藜芦醇为底物刺激木质素过氧化物酶的产生等。总体而言, 与传统碳氮源相比, 以食品残渣为底物时更有利于菌体的生长以及木质纤维素相关酶的产生, 可降低微生物菌剂的生产成本, 具备一定的工业化应用潜力。
2.2 接种量对堆肥过程中木质纤维素降解的影响
木质纤维素降解在堆肥的碳素转化过程中起关键作用, 半纤维素/纤维素降解形成的单糖或寡糖是微生物代谢的主要碳源, 木质素降解形成的多酚是构成腐殖质的重要前体[31]。其中半纤维素为木质纤维素组分中最易降解部分。在本研究中, 半纤维素的降解发生于整个堆肥过程中(图1A), 接种处理(2%IM、4%IM、8%IM)加快了堆肥前期(7 d)半纤维素的降解, 这与接种的菌剂具有较高的木聚糖酶活性有关, 堆肥结束后, 各处理半纤维素的降解率为73.3%~78.6%。纤维素降解规律同半纤维素降解规律相似, 如图1B所示, 接种菌剂加速了整个堆肥过程中纤维素的降解, 堆肥结束时, 2%IM、4%IM、8%IM处理的纤维素降解率分别为66.6%、63.5%、69.3%, 显著高于CK (56.3%)处理(P<0.05); 接种菌剂可有效加快纤维素的降解, 但接种量与纤维素降解率之间并未呈现一致性。木质素是一种复杂的芳香族聚合物, 是堆肥过程中最难降解的有机质组分[27], 如图1C所示, 堆肥过程木质素的降解主要发生于堆肥后期, 这主要由于前期微生物会优先利用易降解有机质组分; 经过35 d的堆肥处理后, 各处理的木质素降解率分别为38.1% (CK)、44.7% (2%IM)、52.6% (4%IM)、66.2% (8%IM), 接种处理显著增加了木质素的降解(P<0.05), 且接种量越高, 木质素降解率越高。总体来讲, 在本研究中, 接种菌剂对促进木质纤维素总量的降解具有明显的正向效应(图1D), 木质纤维素总降解率可达58.7%~72.1%, 其中2%IM、4%IM、8%IM处理木质纤维素的降解率较CK分别提高6.3%、9.2%、23.0% (P<0.05), 木质纤维素总降解率随着接种量的增加而逐步提高。
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图 1 接种量对堆肥过程木质纤维素降解的影响CK: 对照; 2%IM: 添加2%菌剂; 4%IM: 添加4%菌剂; 8%IM: 添加8%菌剂; 不同小写字母表示不同处理间存在显著性差异(P<0.05)。CK: control; 2%IM: adding 2% microbial inoculum; 4%IM: adding 4% microbial inoculum; 8%IM: adding 8% microbial inoculum. Different lowercase letters indicate significant differences among different treatments (P<0.05)Figure 1. Effects of inoculum size on lignocellulose degradation during composting2.3 接种量对堆肥过程中水溶性碳组分的影响
可溶性有机碳(DOC)含量与堆肥稳定性和微生物活性密切相关[32]。堆肥过程中, 大分子有机质经水解作用形成DOC, 水解形成的DOC可被微生物直接吸收利用以进行自身的繁殖代谢。如图2a所示, 在堆肥前期, DOC随接种量的增加而下降, 这是由于接种增加了前期微生物活性, 导致大量DOC被微生物消耗利用, 这与Duan等[33]的研究结果相一致; 而随着堆肥的进行, DOC含量逐渐下降并趋于稳定, 堆肥结束时, 各处理DOC含量稳定在13 g∙kg−1左右, 达到堆肥稳定化标准(<17 g∙kg−1)[24]。
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图 2 接种量对堆肥过程水溶性碳组分的影响CK: 对照; 2%IM: 添加2%菌剂; 4%IM: 添加4%菌剂; 8%IM: 添加8%菌剂。CK: control; 2%IM: adding 2% microbial inoculum; 4%IM: adding 4% microbial inoculum; 8%IM: adding 8% microbial inoculum.Figure 2. Effects of inoculum size on water-soluble carbon components during composting还原糖与多酚作为木质纤维素降解主要产物, 是构成腐殖质的重要前体, 同时是微生物可利用的重要能源物质[34]。多酚与还原糖含量变化如图2b和2c所示, 堆肥前期, 二者含量在各处理中均下降, 这主要由于前期微生物活性较强, 导致过多的多酚与还原糖被降解利用[13]; 而在堆肥后期, 8%IM处理的多酚与还原糖含量明显低于其他处理, 这与其较高的木质纤维素降解现象相矛盾, 主要原因可能在于8%IM处理的微生物活性较高, 导致多酚与还原糖被完全矿化生成了CO2, 这与CO2排放规律相符合。
2.4 接种量对堆肥过程中碳素矿化的影响
堆肥是涉及有机质(OM)矿化与腐殖化相结合的过程, OM含量是反映堆肥产品质量的重要指标[24], OM含量的下降主要因矿化作用所导致。如图3a所示, 4个处理的初始OM含量均在85%左右, 随着堆肥的进行, OM含量逐渐下降, 最终CK、2%IM、4%IM、8%IM处理的OM分别下降至72.6%、71.6%、74.2%和66.8%; 其中8%IM处理的OM含量显著低于其他处理(P<0.05), 表明接种量较高时会加速OM的矿化, 从而增加OM的损失, 这在Duan等[33]研究中也有类似的报道; 而4%IM处理的OM含量较CK处理有所增高, 这与其腐殖化作用较强有关, 从而使碳素得到了固存。
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图 3 接种量对堆肥过程中碳素矿化的影响CK: 对照; 2%IM: 添加2%菌剂; 4%IM: 添加4%菌剂; 8%IM: 添加8%菌剂。CK: control; 2%IM: adding 2% microbial inoculum; 4%IM: adding 4% microbial inoculum; 8%IM: adding 8% microbial inoculum.Figure 3. Effect of inoculum size on carbon mineralization during compostingCO2排放量是反映有机质矿化程度的直接标准[35], 一般来讲, 堆肥初期易降解有机质优先被微生物降解利用, 从而引起CO2的大量排放[36], 本研究的试验现象也符合这一规律(图3b); 而到堆肥后期, 难降解有机质成分占主导地位, 微生物活性下降, CO2排放逐渐趋于稳定, 然而本研究中8%IM处理堆肥后期的CO2排放量同样出现明显增加趋势, 表明过量接种同样会增加难降解OM的矿化程度; 堆肥结束时, 8%IM处理的CO2累积排放量相较于CK、2%IM、4%IM分别增加21.9%、22.3%、26.0%, 这与OM含量的变化相符合。
2.5 接种量对堆肥过程中腐殖化过程的影响
腐殖化是堆肥过程中固定碳素的重要途径, 腐殖质(HS)含量决定了堆肥产品质量和应用潜力[37], HS主要包含大分子腐殖酸(HA)和小分子富里酸(FA)两种组分。如图4a所示, 堆肥过程中各处理HS含量均呈现先下降后缓慢增长的现象, 这主要是由于初始原料中的HS多为小分子的FA组分, 在堆肥高温期被微生物大量降解利用[13], 而到堆肥后期, 小分子物质通过不同途径聚合形成HA, 引起HS含量的上升; 堆肥结束时, CK、2%IM、4%IM、8%IM处理的HS含量分别为103.5 g∙kg−1、107.6 g∙kg−1、117.2 g∙kg−1、89.3 g∙kg−1, 其中4%IM处理的HS含量最高, 表明适当接种更有助于HS的形成和保存。
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图 4 接种量对堆肥过程中腐殖化的影响CK: 对照; 2%IM: 添加2%菌剂; 4%IM: 添加4%菌剂; 8%IM: 添加8%菌剂。CK: control; 2%IM: adding 2% microbial inoculum; 4%IM: adding 4% microbial inoculum; 8%IM: adding 8% microbial inoculum.Figure 4. Effect of inoculum size on humification during compostingFA的动态变化如图4b所示。各处理初始FA含量在90 g∙kg−1左右, 并在堆肥前14 d内被大量降解, 最终稳定在25 g∙kg−1左右。与FA变化规律相反, HA含量在堆肥后期逐渐上升(图4c), 其中4%IM处理的HA含量最终达91.3 g∙kg−1, 相较于CK、2%IM和8%IM处理分别提高了24.9%、10.7%和35.8%, 接种一定量的菌剂加速了木质纤维素降解形成腐殖酸前体物并聚合形成HA, 而过量接种(8%IM)虽更能增加木质纤维素的降解, 然而形成的前体物更多地被微生物完全矿化生成了CO2, 多酚、还原糖以及CO2的变化规律也证实了这一观点。
腐殖化指数(HI)、聚合度(DP)和腐殖酸占比(PHA)能够准确地反映堆肥腐殖化程度[13], 其中, HI代表HA占TOC的比例, HI的上升表明有机碳发生了强烈的腐殖化作用[12]。堆肥结束时, 2%IM (19.9%)、4%IM (21.2%)处理的HI显著高于CK (17.4%)和8%IM(17.4%)处理(P<0.05), 且以4%IM处理最佳(图4d), 说明4%接种更有利于碳素向腐殖化过程的流动; DP、PHA分别代表HA/FA与HA/HS的比值, DP、PHA的增加象征着HS组分复杂性与稳定性的增强[12]。与CK相比, 接种处理(2%IM、4%IM、8%IM)的DP、PHA值均显著提高(P<0.05) (图4e, f)。这些结果表明接种更有助于堆肥产品的成熟性和稳定性。
2.6 接种量对堆肥碳素转化途径的影响
本研究利用偏最小二乘路径模型对堆肥过程碳素转化路径进行了更加直观清晰的分析, 如图5所示。接种菌剂增加了木质纤维素(Lce)对DOC的影响, 表明接种有利于木质纤维素降解形成DOC, 且接种量越高, 影响效果越明显; 而在DOC向HA和CO2的转化途径中, 8%IM处理DOC与CO2的相关性更高(r=−0.93), 进一步说明过量接种引起较高的微生物代谢活性, 微生物为维持自身的代谢而分解利用HA前体, 加速了碳的完全矿化; 而4%IM处理能够提高DOC向HA的定向转化, 增加堆肥中HA的含量; 此外, CK处理中DOC向HA转化同样具有较高的相关系数(−0.87), 这说明常规堆肥过程中木质纤维素降解产物可有效进入到腐殖化途径, 而木质纤维素降解率是影响腐殖化进行的限制因素。由此可见, 接种微生物有利于破除因木质纤维素降解困难而导致堆肥腐殖化程度较低的难题, 而接种量会影响微生物菌剂功能的有效发挥, 适量接种有助于Lce向HA的定向转化。
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图 5 接种量对堆肥碳素转化途径的影响DOC: 可溶性有机碳; Lce: 木质纤维素; HA: 腐殖酸。CK: 对照; 2%IM: 添加2%菌剂; 4%IM: 添加4%菌剂; 8%IM: 添加8%菌剂。DOC: dissolved organic carbon; Lce: lignocellulose; HA: humic acid. CK: control; 2%IM: adding 2% microbial inoculum; 4%IM: adding 4% microbial inoculum; 8%IM: adding 8% microbial inoculum.Figure 5. Effects of inoculum size on carbon conversion pathway during composting3. 讨论
3.1 接种量对堆肥木质纤维素组分降解的差异分析
好氧堆肥的实质为微生物主导的碳氮转化过程, 微生物作为主要的驱动者, 其代谢活性决定了碳素的转化效率和方向[38]。大量研究表明, 接种外源微生物可促进木质纤维素的降解[9-11], 本研究发现接种菌剂对堆肥过程中半纤维素、纤维素、木质素的降解呈现不同的影响效果。接种菌剂并未明显提高半纤维素的降解率, 这主要由于半纤维素结构简单, 易被微生物降解利用[27], 而经过35 d的好氧堆肥处理后, 堆体中的土著微生物可完全将半纤维素降解至稳定水平。Serramiá等[39]在进行橄榄废弃物堆肥过程中也发现半纤维素的降解主要发生在前6周, 随后降解逐渐变缓并趋于稳定。因此, 无论是否接种, 堆肥过程中半纤维素均具有较高的降解水平, 接种菌剂仅是加快了前期半纤维素降解速率, 并未影响半纤维素的降解总量。而针对纤维素来讲, 接种菌剂可有效加快纤维素的降解, 但接种量对纤维素的降解并无明显影响。这可能由于青霉菌和曲霉菌具有较高的纤维素降解能力[40], Tian等[41]在进行中药渣堆肥试验时也发现曲霉菌在纤维素的降解过程中起主要作用; 同时本研究在利用食品残渣进行菌种扩繁后, 活菌数与酶活性均有了明显提高; 因此, 仅需少量接种即可将纤维素降解至稳定水平。接种量与木质素的降解呈现正相关关系, 表明堆体土著微生物对木质素的降解作用较弱, 接种功能菌剂可增加木质素降解相关微生物数量和酶活, 加快堆肥过程木质素的降解, 这在Hu等[42]的堆肥研究中均得到证实。
因此, 接种量对各组分降解的影响效果为: 木质素>纤维素>半纤维素, 且接种量越大, 对难降解组分的降解促进效果越为明显, 表明外源微生物接种更侧重于完成堆体土著微生物难以完成的工作, 然而其前提是需保证外源接种的微生物对木质纤维素各组分均具有一定的降解能力。前人研究中也发现相似的现象, Hu等[42]在菌渣堆肥过程接种木质纤维素降解菌群, 发现半纤维素、纤维素和木质素的降解率分别提高8.8%、19.6%和34.5%, 接种复合菌剂对木质素的降解效果提升更加明显。
3.2 接种量对堆肥碳素转化途径的差异分析
堆肥过程中菌剂接种量一般在0.05%~5%, 最高可达10%[16], 这取决于菌剂的质量、接种时间以及堆肥物料的类型。接种量对碳素转化的影响至关重要, 接种量过低不利于外源菌剂的定殖从而使其难以发挥相应功能, 接种量过高有助于提高微生物代谢, 然而较高的微生物活性易引起有机质的过量矿化, 降低堆肥腐熟程度[43]。Duan等[33]在探讨不同枯草芽孢杆菌(Bacillus subtilis)接种量(0.5%、1%、2%)对牛粪堆肥过程中的碳素转化的影响发现, 过量接种(2%)加速了CO2的排放, 不利于碳素的固持和腐殖化的进行; 劳德坤等[44]探讨了不同复合菌剂接种量(1%、3%、5%)对蔬菜废弃物堆肥的影响, 发现接种量为3%时堆肥腐熟程度最高。而本研究探讨了不同真菌(青霉菌、曲霉菌)接种量对园林废弃物堆肥碳素转化的影响, 过量接种标准(8%)与前人研究结果存在一定差异, 除因菌种不同外, 主要由于本研究堆肥原料为园林废弃物, 其惰性有机质成分含量较高, 因此需要较高的接种量才可达到相应效果。本研究结果表明4%接种量有助于园林废弃物堆肥过程中木质纤维素向HA的定向转化。但不同菌剂针对不同废弃物堆肥的接种标准有待进一步研究, 本研究为优化园林废弃物堆肥接种工艺、提高HA含量和减少CO2排放提供理论支持。
4. 结论
本研究利用食品残渣(苹果渣、豆渣)扩繁木质纤维素降解菌, 扩繁产品活菌数可达3.7×1010 cfu∙mL−1, 符合GB 20287—2006《农用微生物菌剂》的标准(>109 cfu∙mL−1), 活菌数较常规培养基显著增加了46.2% (P<0.05), 且木质纤维素降解酶活性较常规培养基也均有显著提高(P<0.05)。将扩繁菌剂接种于园林废弃物堆肥中可有效加速木质纤维素的降解, 其中, 8%接种量(8%IM)最有利于木质纤维素的降解(降解率为72.14%), 但同时也增加了碳素的矿化, 不利于腐殖化作用的进行; 而4%IM处理加快木质纤维素降解形成腐殖酸前体物的同时促进了腐殖酸(91.3 g∙kg−1)的形成, 减少了堆肥过程碳素损失, 并提高了堆肥产品腐熟程度。本研究为园林废弃物高效堆肥以及多源废弃物的协同处理提供理论依据。
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图 1 接种量对堆肥过程木质纤维素降解的影响
CK: 对照; 2%IM: 添加2%菌剂; 4%IM: 添加4%菌剂; 8%IM: 添加8%菌剂; 不同小写字母表示不同处理间存在显著性差异(P<0.05)。CK: control; 2%IM: adding 2% microbial inoculum; 4%IM: adding 4% microbial inoculum; 8%IM: adding 8% microbial inoculum. Different lowercase letters indicate significant differences among different treatments (P<0.05)
Figure 1. Effects of inoculum size on lignocellulose degradation during composting
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图 2 接种量对堆肥过程水溶性碳组分的影响
CK: 对照; 2%IM: 添加2%菌剂; 4%IM: 添加4%菌剂; 8%IM: 添加8%菌剂。CK: control; 2%IM: adding 2% microbial inoculum; 4%IM: adding 4% microbial inoculum; 8%IM: adding 8% microbial inoculum.
Figure 2. Effects of inoculum size on water-soluble carbon components during composting
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图 3 接种量对堆肥过程中碳素矿化的影响
CK: 对照; 2%IM: 添加2%菌剂; 4%IM: 添加4%菌剂; 8%IM: 添加8%菌剂。CK: control; 2%IM: adding 2% microbial inoculum; 4%IM: adding 4% microbial inoculum; 8%IM: adding 8% microbial inoculum.
Figure 3. Effect of inoculum size on carbon mineralization during composting
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图 4 接种量对堆肥过程中腐殖化的影响
CK: 对照; 2%IM: 添加2%菌剂; 4%IM: 添加4%菌剂; 8%IM: 添加8%菌剂。CK: control; 2%IM: adding 2% microbial inoculum; 4%IM: adding 4% microbial inoculum; 8%IM: adding 8% microbial inoculum.
Figure 4. Effect of inoculum size on humification during composting
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图 5 接种量对堆肥碳素转化途径的影响
DOC: 可溶性有机碳; Lce: 木质纤维素; HA: 腐殖酸。CK: 对照; 2%IM: 添加2%菌剂; 4%IM: 添加4%菌剂; 8%IM: 添加8%菌剂。DOC: dissolved organic carbon; Lce: lignocellulose; HA: humic acid. CK: control; 2%IM: adding 2% microbial inoculum; 4%IM: adding 4% microbial inoculum; 8%IM: adding 8% microbial inoculum.
Figure 5. Effects of inoculum size on carbon conversion pathway during composting
表 1 试验材料的理化性状
Table 1 Physical and chemical properties of the experimental materials
试验材料
Material含水率
Moisture
content (%)有机质a
Organic
matter (%)总氮a
Total
nitrogen (%)碳氮比a
C/NpH 电导率
Electrical
conductivity (mS·cm−1)总糖a
Total sugar
content (%)园林废弃物
Garden waste11.00±0.34 84.74±0.56 1.49±0.00 34.14±0.20 7.32±0.02 1.55±0.17 — 餐厨垃圾
Food waste78.55±2.23 91.74±0.11 2.89±0.02 14.61±0.32 4.58±0.07 3.30±0.20 — 苹果渣
Apple pomace82.81±0.58 98.04±0.14 0.81±0.02 69.87±1.82 5.67±0.09 — 20.75±0.58 豆渣
Bean dregs79.95±4.14 95.73±0.07 2.64±0.01 21.02±0.09 7.07±0.04 — 37.29±1.29 “a”: 基于物料干重; “—”: 表示未检测。“a”: based on dry weight of material; “—”: not measured. 
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表 2 食品残渣(苹果渣、豆渣)代替常规碳、氮源(培养基 Ⅱ)对培养72 h后菌液活菌数与酶活性的影响
Table 2 Effect of food residues (apple pomace, soybean dregs) instead of conventional carbon and nitrogen sources (Medium Ⅱ) on the viable count and enzymatic activity of the broth after 72 h incubation
处理
Treatment活菌数
Viable count
(×010 cfu∙mL−1)木聚糖酶
Xylanase
(U∙mL−1)纤维素酶
Cellulase
(U∙mL−1)漆酶
Laccase
(U∙mL−1)锰过氧化物酶
Manganese peroxidase
(U∙mL−1)木质素过氧化物酶
Lignin peroxidase
(U∙mL−1)培养基Ⅰ Medium Ⅰ 2.53±0.41a 337.40±108.81a 73.82±9.55a 3.71±0.69a 11.26±3.97a 2.98±1.67a 培养基Ⅱ Medium Ⅱ 3.70±0.37b 597.40±171.21b 99.47±5.99b 4.91±2.22a 22.38±2.46b 12.39±1.67b 不同小写字母表示不同处理间存在显著性差异(P<0.05)。Different lowercase letters indicate significant differences between two treatments (P<0.05).
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