油茶壳堆肥微生物的培养及功能芽孢杆菌的筛选和初步应用

张声峰; 陈硕昌; 梁文凤; 严祥; 冯艺峰; 李桂珍; 杨辉

doi:10.19303/j.issn.1008-0384.2021.07.014

油茶壳堆肥微生物的培养及功能芽孢杆菌的筛选和初步应用

doi: 10.19303/j.issn.1008-0384.2021.07.014

1.
广西大学生命科学与技术学院，广西　南宁　530004
2.
广西壮族自治区林业科学研究院，广西　南宁　530000

基金项目: 广西科技重大专项（桂科 AA20302021-7）；百色市科学研究与技术开发计划（百科20192129）；广西特色经济林培育与利用重点实验室自主课题（JA-20-04-02）

详细信息

作者简介:
张声峰（1990−），男，硕士研究生，研究方向：微生物工程（E-mail：1908391037@st.gxu.edu.cn）

通讯作者:
杨辉（1972−），男，博士，高级工程师，研究方向：食品发酵和酶工程（E-mail：huiy422@gxu.edu.cn）

中图分类号: X 712；X 53
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- 被引次数: 0
出版历程
- 收稿日期: 2021-03-02
- 修回日期: 2021-04-25
- 网络出版日期: 2021-07-13
- 刊出日期: 2021-07-28

Functional Bacillus Species in Camellia Seed Shell Compost

1.
College of Life Science and Technology, Guangxi University, Nanning, Guangxi　530004, China
2.
Guangxi Academy of Forestry Sciences, Nanning, Guangxi　530000, China

摘要

摘要: 目的从自然状态下的油茶壳堆肥产品中筛选功能芽孢杆菌，为油茶壳堆肥高效生产、饲料发酵等应用提供菌剂。方法利用高通量测序技术测定广西某油茶壳堆肥中微生物群落分布，筛选适宜堆肥微生物生长的培养基以获得多样性较丰富的堆肥芽孢杆菌菌群，经平板稀释涂布法分离纯化菌株，使用水解圈法和酶活测定方法进行水解酶功能分析，利用菌株形态特征观察、16S rDNA分子鉴定法明确菌株的种属，并运用分子生物学软件 MEGAX构建系统发育树。使用重铬酸钾法测定腐殖酸含量。结果芽孢杆菌科（Bacillaceae）是优势菌科，占比达55.58%；分离到15株芽孢杆菌，同时具有淀粉酶、纤维素酶、蛋白酶3种水解酶活性的菌株有6株；同时具有淀粉酶和纤维素酶活性的有1株；仅具有蛋白酶或纤维素酶一种酶活性的有2株。其中菌株Bacillus sp. YX11的蛋白酶活力达到27.07±3.28 U·mL⁻¹，淀粉酶活力达123.97±3.19 U·mL⁻¹，纤维素酶活力达15.75±0.23 U·mL⁻¹。含有这15株菌的复合菌制剂有利于腐殖酸的生成，提高了堆肥品质。结论鉴定出的Bacillus cereus YX02和 Bacillus flexus FYF01等菌株具有进一步研究开发的价值，可用于开发油茶壳堆肥微生物菌制剂。
- 油茶壳堆肥 /
- 芽孢杆菌 /
- 功能性 /
- 筛选 /
- 应用
Abstract: Objective Bacillus spp that contribute to the fermentation of Camellia oleifera seed shells were isolated for effective composting of the waste material. Method The microbial community in natural camellia seed shell compost found in Guangxi was studied using the high-throughput sequencing technology. Suitable culture medium to foster the growth of richly diverse Bacillus spp from the compost was selected. Flora isolation by dilution with a streaking plate method followed. The hydrolase activities of the isolates were determined by using the hydrolysis circle method and enzyme activity analysis, the species identified by a 16S rDNA analysis, and the phylogeny constructed by MEGAX. The content of humic acid in the compost was measured by a potassium dichromate method. Result Bacillaceae was the dominant family in the compost. It accounted for 55.58% of all isolated flora. Among the 15 Bacillus isolates, 6 exhibited activities of amylase, cellulase, and protease, one of amylase and cellulase, and 2 of protease or protease. Strain YX11 showed a protease activity of 27.07±3.28 U·mL⁻¹, an amylase activity of 123.97±3.19 U·mL⁻¹, and a cellulase activity of 15.75±0.23 U·mL⁻¹. In the presence of numerous Bacillus spp that secreted varieties of hydrolases, formation of humic acid in the compost was enhanced. Conclusion Some of the isolated strains, such as B. cereus YX02 and B. flexus FYF01, might warrant further investigation to develop microbial inoculants for efficient composting camellia seed shells.
- camellia seed shell compost /
- bacillus /
- functional /
- screening /
- application

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图 1 油茶壳堆肥原核微生物群落组成（科类）

Figure 1. Composition of prokaryotic microbial community at family level in camellia seed shell compost

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图 2 六组培养基中的细菌数量和OD₆₀₀值的变化趋势

注：Y：厌氧培养，HJ：好氧静置培养，HY：好氧摇床培养

Figure 2. Bacteria CFU by plate count and OD₆₀₀ measurement on 6 culture media

Notes: Y: anaerobic culture; HJ: aerobic static culture; HY: aerobic culture on shaking table.

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图 3 光学显微镜下部分菌株革兰氏染色图（×4 000倍）

注：a：菌株YX07；b：菌株FYF01；c：菌株YX12；d：菌株FYF07

Figure 3. Gram staining bacteria morphology under microscope (×4 000)

Notes: a: Strain YX07; b: Strain FYF01; c: Strain YX12; d: Strain FYF07.

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图 4 16S rDNA序列PCR产物琼脂凝胶电泳结果

注：M：DL2000 （DNA Marker）；1~8：部分菌株基因组 PCR产物

Figure 4. Agarose gel electrophoresis of PCR products from 16S rDNA sequence

Notes: M: DL2000 (DNA marker); 1-8: genomic PCR products of strains.

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图 5 基于16S rDNA序列的系统发育树

Figure 5. Phylogenetic tree of strains based on 16S rDNA sequence

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图 6 菌株的蛋白酶、淀粉酶、纤维素酶活力

Figure 6. Activities of protease, amylase, and cellulase of strains

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图 7 应用试验腐殖酸含量

Figure 7. Humic acid content in compost at application trial

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表 1 菌制剂应用试验配方

Table 1. Proposed formulations of microbial inoculants

组别 Group	油茶壳 Camellia shell/g	菌液 microbial inoculants/mL	糖蜜 Molasses/mL	花生枯 Peanut cake/g	尿素 Urea/g	水 H₂O/mL
菌制剂 experiment group	15	15	6	1.2	1.2	100
对照组 control group	15	0	6	1.2	1.2	115

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表 2 菌株形态特征

Table 2. Morphological characteristics of isolated Bacillus spp

菌株编号 Strains	菌落质地 Colony texture	菌落颜色 Colony color	菌落形态 Colony morphology
YX01	干燥、皱褶 dry and wrinkled	半透明色 translucent color	不规则圆形，有毛边 irregular round with rough edges
YX02	干燥、皱褶 dry and wrinkled	乳白色 milky white	较规则圆形，中间隆起 regular round, middle uplift
YX03	光滑、湿润 smooth and moist	半透明色 translucent color	较规则圆形 regular round
YX07	光滑、湿润 smooth and moist	乳白色 milky white	圆形 round
YX08	光滑、湿润 smooth and moist	半透明色 translucent color	圆形 round
YX11	湿润、黏稠 wet and sticky	乳白色 milky white	圆形 round
YX12	光滑、湿润 smooth and moist	乳白色 milky white	圆形 round
YX13	干燥、皱褶 dry and wrinkled	枯黄色 withered yellow	不规则圆形，有毛边 irregular round with rough edges
YX15	光滑、湿润 smooth and moist	乳白色 milky white	圆形 round
YX16	光滑、湿润 smooth and moist	乳白色 milky white	圆形 round
FYF01	干燥、皱褶 dry and wrinkled	乳白色 milky white	不规则圆形，有毛边 irregular round with rough edges
FYF04	光滑、湿润 smooth and moist	乳白色 milky white	较规则圆形 regular round
FYF07	光滑、湿润 smooth and moist	乳白色 milky white	较规则圆形，中间隆起 regular round, middle uplift
FYF08	干燥、皱褶 dry and wrinkled	半透明色 translucent color	不规则圆形，有毛边 irregular round with rough edges
FYF10	光滑、湿润 smooth and moist	乳白色 milky white	较规则圆形 regular round

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表 3 菌株16S rDNA鉴定结果

Table 3. Identifications by 16S rDNA

序号 Serial number	编号 Number	菌种分类 Species classification
1	YX01	Bacillus sp.
2	YX02	Bacillus cereus
3	YX03	Bacillus sp.
4	YX07	Bacillus sp.
5	YX08	Bacillus sp.
6	YX11	Bacillus sp.
7	YX12	Bacillus sp.
8	YX13	Bacillus sp.
9	YX15	Brevibacterium oceani
10	YX16	Bacillus sp.
11	FYF01	Bacillus flexus
12	FYF04	Bacillus sp.
13	FYF07	Bacillus sp.
14	FYF08	Bacillus sp.
15	FYF10	Bacillus sp.

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表 4 不同培养基上菌落直径与透明圈直径比

Table 4. Ratio of transparent ring diameter to colony diameter on different mediums

菌株编号 Strains	脱脂乳糖培养基 Skim milk medium			淀粉培养基 Starch medium			刚果红培养基 Congo red medium
菌株编号 Strains	透明圈直径D Transparent ring diameter/mm	菌落直径d Colony diameter/mm	直径比D/d Diameter ratio	透明圈直径D Transparent ring diameter/mm	菌落直径d Colony diameter/mm	直径比D/d Diameter ratio	透明圈直径D Transparent ring diameter/mm	菌落直径d Colony diameter/mm	直径比D/d Diameter ratio
YX01	0.0	7.5	0.00	13.0	7.5	1.73	18.0	5.0	3.60
YX02	28.0	26.5	1.06	0.0	16.0	0.00	0.0	11.0	0.00
YX03	0.0	0.0	0.00	0.0	4.0	0.00	20.0	8.0	2.50
YX07	0.0	0.0	0.00	0.0	6.5	0.00	0.0	8.5	0.00
YX08	0.0	0.0	0.00	0.0	11.0	0.00	0.0	2.0	0.00
YX11	25.0	20.0	1.25	19.5	6.0	3.25	30.0	11.0	2.73
YX12	0.0	0.0	0.00	0.0	5.0	0.00	0.0	8.0	0.00
YX13	29.0	23.0	1.26	20.0	8.0	2.50	18.0	8.5	2.12
YX15	0.0	0.0	0.00	0.0	6.5	0.00	0.0	10.0	0.00
YX16	0.0	0.0	0.00	0.0	4.0	0.00	0.0	7.0	0.00
FYF01	14.0	12.0	1.17	16.0	6.0	2.67	32.0	6.5	4.92
FYF04	30.0	21.5	1.39	14.0	7.0	2.00	24.5	6.0	4.08
FYF07	26.0	19.0	1.37	20.0	11.5	1.74	24.0	9.0	2.67
FYF08	0.0	0.0	0.00	0.0	7.0	0.00	0.0	2.0	0.00
FYF10	25.5	19.5	1.31	18.0	12.5	1.44	25.0	9.0	2.78

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