3.2 Development of Complex Microbial Inoculants
67
bacterium suspension was added into 1000 mL of PCS medium for conducting subculture, which was continuously carried out for ten generations in order to stabilize
the microbial community structure.
Isolation and identification of complex microbial inoculants: By using spread plate
method, 20 strains of bacteria and 10 trains of fungi were separately screened from
the inoculation agent. By using boiling method, minor DNA was rapidly extracted
from bacteria for polymerase chain reaction (PCR) amplification while genomic
DNA of fungi was extracted by employing DNA extraction kit. Furthermore, 5 µL
of extracting solution for genome of fungi was taken and detected by applying 1% of
agarose gel electrophoresis (AGE), showing a clear and bright band, indicating that
no degradation appeared. Therefore, PCR amplification of 28S rDNA gene can be
conducted. After conducting PCR amplification of 16S rDNA gene, target fragments
of 16S rDNA genes of 20 strains of bacteria were acquired, with the size about 1600 bp
obtained through AGE, as shown in Fig. 4.5. After carrying out PCR amplification of
28S rDNA genes, only the target fragments of 28S rDNA gene of a strain of fungus
were attained, with the size about 700 bp detected through AGE.
The purified PCR products were sequenced and the result is displayed in Table
3.1. To be specific, 20 strains of bacteria were divided into eight types, including
Aeribacillus sp., Bacillus licheniformis, Bacillus thermoamylovorans, Brevibacillus
borstelensis, Geobacillus sp., Lysinibacillus sp., Paenibacillus dendritiformis and
Ureibacillus thermosphaericus, all belonging to Bacillales. The fungus Penicillium
sp. belongs to Eurotiomycetes.
3.2.2 Acidification-Resistant Complex Microbial Inoculants
Culture of complex microbial inoculants: During inhibitory stage of acidification
of kitchen waste compost, some samples were taken to prepare bacterium suspension. By using limiting culture and oriented acclimatization generation of culture
medium of mixed acids [m (acetic acid: propionic acid: butyric acid: lactic acid)],
acidification-resistant complex microbial inoculants capable of efficiently degrading
micromolecular organic acids with were prepared. The culture was conducted using
complete medium (50 mL), and the proportion of mixed acids was 3:3:2.5:12.5 and
pH was adjusted to 5.3, in which 1 mL of bacterium suspension was added. The
mixture was cultured at the constant temperature of 50 °C for 24 h at the rotational
speed of 200 r/min. According to the aforementioned steps, subculture was carried
out in which addition amount of mixed acids increased step by step. After continuously culturing 50 generations, microbial community structure was stable, that is,
acquiring a complex microbial system for degrading organic acids.
Isolation and identification of complex microbial inoculants: The SEM images,
colonial morphology and characteristics of seven strains of single bacteria isolated
from acidification-resistant complex microbial inoculants are shown in Fig. 3.3. It
can be seen from the Fig. 3.3 and Table 3.2 that round light yellow S-1 bacterial
colony with the diameter about 5 mm was transparent, whose surface was moist,
67
bacterium suspension was added into 1000 mL of PCS medium for conducting subculture, which was continuously carried out for ten generations in order to stabilize
the microbial community structure.
Isolation and identification of complex microbial inoculants: By using spread plate
method, 20 strains of bacteria and 10 trains of fungi were separately screened from
the inoculation agent. By using boiling method, minor DNA was rapidly extracted
from bacteria for polymerase chain reaction (PCR) amplification while genomic
DNA of fungi was extracted by employing DNA extraction kit. Furthermore, 5 µL
of extracting solution for genome of fungi was taken and detected by applying 1% of
agarose gel electrophoresis (AGE), showing a clear and bright band, indicating that
no degradation appeared. Therefore, PCR amplification of 28S rDNA gene can be
conducted. After conducting PCR amplification of 16S rDNA gene, target fragments
of 16S rDNA genes of 20 strains of bacteria were acquired, with the size about 1600 bp
obtained through AGE, as shown in Fig. 4.5. After carrying out PCR amplification of
28S rDNA genes, only the target fragments of 28S rDNA gene of a strain of fungus
were attained, with the size about 700 bp detected through AGE.
The purified PCR products were sequenced and the result is displayed in Table
3.1. To be specific, 20 strains of bacteria were divided into eight types, including
Aeribacillus sp., Bacillus licheniformis, Bacillus thermoamylovorans, Brevibacillus
borstelensis, Geobacillus sp., Lysinibacillus sp., Paenibacillus dendritiformis and
Ureibacillus thermosphaericus, all belonging to Bacillales. The fungus Penicillium
sp. belongs to Eurotiomycetes.
3.2.2 Acidification-Resistant Complex Microbial Inoculants
Culture of complex microbial inoculants: During inhibitory stage of acidification
of kitchen waste compost, some samples were taken to prepare bacterium suspension. By using limiting culture and oriented acclimatization generation of culture
medium of mixed acids [m (acetic acid: propionic acid: butyric acid: lactic acid)],
acidification-resistant complex microbial inoculants capable of efficiently degrading
micromolecular organic acids with were prepared. The culture was conducted using
complete medium (50 mL), and the proportion of mixed acids was 3:3:2.5:12.5 and
pH was adjusted to 5.3, in which 1 mL of bacterium suspension was added. The
mixture was cultured at the constant temperature of 50 °C for 24 h at the rotational
speed of 200 r/min. According to the aforementioned steps, subculture was carried
out in which addition amount of mixed acids increased step by step. After continuously culturing 50 generations, microbial community structure was stable, that is,
acquiring a complex microbial system for degrading organic acids.
Isolation and identification of complex microbial inoculants: The SEM images,
colonial morphology and characteristics of seven strains of single bacteria isolated
from acidification-resistant complex microbial inoculants are shown in Fig. 3.3. It
can be seen from the Fig. 3.3 and Table 3.2 that round light yellow S-1 bacterial
colony with the diameter about 5 mm was transparent, whose surface was moist,