**3. Materials and methods**

Fresh Spent Mushroom Substrate (SMS) of *Pleurotus eous* was used for the present study. SMS was obtained by growing *Pleurotus eous* mushroom using paddy straw in the mushroom house at Post Graduate and Research Department of Botany, Thiagarajar College, Madurai. The spent paddy straw substrate after three yields of the mushroom *P. eous* was used fresh for characterization studies, dried in the sun and powdered for use in further analyses.

**251**

*Screening and Potential Uses of Contaminated Spent Mushroom (*Pleurotus *spp.)*

For pot culture studies, the powdered SMS was mixed with garden soil in different

Twenty seeds of green gram and black gram, fifty seeds of tomato and chili were sown in each pot containing garden soil with SMS in various concentrations. Pot contained only the garden soil was maintained as control for each treatment. The treatments were laid out in a completely randomized pattern with three replicates per treatment. Germination was performed under ambient conditions in the net house, and pots were irrigated daily. Growth was monitored up to 30 days after

Sample of SMS of *Pleurotus eous* was mechanically stirred with distilled water. The SMS suspension was clarified by centrifugation, serially diluted (10−2 to 10−7) with sterile water and inoculated on potato dextrose agar medium (PDA) and nutrient agar (NA) medium. All the strains observed growing on plates were arbitrarily selected, transferred and maintained in new PDA or NA plates for further use. A representative sample colony of each visually differentiable bacterium was selected using a sterile inoculating loop. Each colony was transferred by streaking an inoculating loop in parallel lines over four quadrants of NA plate. The plates were

observations about the shape, color, size and other visual properties of each isolate were recorded. The bacterial strains isolated from the SMS of *Pleurotus eous* were

**3.3 Antifungal activity of bacteria isolated from SMS in dual culture plate assay**

The fungal pathogens viz., *Fusarium sp., Alternaria sp., Phytophthora sp.*, and *Aspergillus sp*., were obtained from Biotechnology lab at Post Graduate and Research

Antifungal activity of bacteria isolated from SMS of *Pleurotus eous* mushroom was evaluated in a dual plate assay against four fungal pathogens of plants viz., *Fusarium sp.*, *Alternaria sp.*, *Phytophthora sp.* and *Aspergillus sp.* The bacteria and fungi were cultured in opposing fashion on PDA plates. Bacterial isolate broth (100 μl) of approximately 108 cells/ml was inoculated on PDA plates using spread plate technique. Mycelial agar plugs of one day old culture of the test fungi and the bacterial isolate were inoculated opposite to each other. All the plates were maintained at room temperature and observed for the appearance of zone of inhibition surrounding the bacterial colony where the fungal mycelium

The empirical data depicted in **Tables 1** and **2** show the composition of Spent Mushroom Substrate (SMS) of *Pleurotus* spp., compared with the substrate, paddy straw. The cellulose and hemicellulose content of paddy straw substrate were 30.25% and 23.18% and dry weight respectively. Cultivation of *Pleurotus eous* on the substrate had lowered the cellulose content by 24.89%. The hemicellulose

characterized and identified by using the standard procedures.

Department of Botany, Thiagarajar College, Madurai.

**4.1 Composition of spent mushroom substrate (SMS)**

c for twenty-four hour. The isolated colonies were used for initial

*DOI: http://dx.doi.org/10.5772/intechopen.93863*

ratio as 20%,40%,60%80%100% and control.

**3.2 Isolation of microbial flora from SMS**

**3.1 Growth studies**

seeding.

incubated at 37o

failed to grow.

**4. Observations and results**

Laboratory tests measured the following properties: PH, EC, soluble salts, moisture, organic matter, carbon, nitrogen and phosphorus, potassium, Carbon:Nitrogen (C:N) ratio, cellulose and hemicelluloses.

*Screening and Potential Uses of Contaminated Spent Mushroom (*Pleurotus *spp.) DOI: http://dx.doi.org/10.5772/intechopen.93863*

#### **3.1 Growth studies**

*Emerging Contaminants*

Barley straw.

*Actinobacteria*.

fungi.

**2.6 Microbial flora of SMS**

Objectives of the study.

involved the followings objectives:

• Isolation of bacteria from SMS.

and powdered for use in further analyses.

**3. Materials and methods**

room substrate (SMS) of *Pleurotus eous*.

phosphorous (0.48%) and sodium (0.29%).

**2.5 Anti-microbial activity of SMS**

(2.26%) and calcium (5.16%) were comparatively higher to the availability of total

The uncontrolled use of antibiotics has caused serious problems in human and animal health, causing that pathogens develop resistance to them, so World Health Organization considered the infections caused by pathogens resistant to drugs as a public health problem; therefore, it is necessary to find new pharmacological strategies, among which we can find natural products such as plants and fungi. The results showed that in the case of *Escherichia coli*, the greatest inhibition zone was of 12.66 mm at a concentration of 6 mg ml−1, with treatment of *Lentinula*

*edodes*/cedar; *Salmonella typhimurium* showed a greatest inhibition zone of

31.10 mm to a concentration of 5.12 mg mL-1, with treatment of *Pleurotus ostreatus*/

The bacterial diversity in SMC by using molecular techniques in order to reveal the origin of SMC microflora and its potential effect on soil microbial communities after incorporation into agricultural soils [30, 34, 35]. Fifty bacterial isolates were classified into 14 operational taxonomic units (OTUs) following ARDRA-PCR of the 16S rDNA gene. Sequencing of the 16S rDNA amplicon assigned 12 of the 14 OTUs to Gram-positive bacteria, associated with the genera *Bacillus*, *Paenibacillus*, *Exiguobacterium*, *Staphylococcus*, *Desemzia*, *Carnobacterium*, *Brevibacterium*, *Arthrobacter* and *Microbacterium* of the bacterial divisions *Firmicutes* and

With the background research and the literature review on spent mushroom substrate (SMS), an attempt was made to isolate, identify and characterize the microbial flora of spent mushroom substrate of *Pleurotus eous*. The research

• Analysis of the composition and physicochemical properties of spent mush-

• Antimicrobial activity of the bacterial isolates against selected soil pathogenic

Fresh Spent Mushroom Substrate (SMS) of *Pleurotus eous* was used for the present study. SMS was obtained by growing *Pleurotus eous* mushroom using paddy straw in the mushroom house at Post Graduate and Research Department of Botany, Thiagarajar College, Madurai. The spent paddy straw substrate after three yields of the mushroom *P. eous* was used fresh for characterization studies, dried in the sun

Laboratory tests measured the following properties: PH, EC, soluble salts,

moisture, organic matter, carbon, nitrogen and phosphorus, potassium,

Carbon:Nitrogen (C:N) ratio, cellulose and hemicelluloses.

**250**

For pot culture studies, the powdered SMS was mixed with garden soil in different ratio as 20%,40%,60%80%100% and control.

Twenty seeds of green gram and black gram, fifty seeds of tomato and chili were sown in each pot containing garden soil with SMS in various concentrations. Pot contained only the garden soil was maintained as control for each treatment. The treatments were laid out in a completely randomized pattern with three replicates per treatment. Germination was performed under ambient conditions in the net house, and pots were irrigated daily. Growth was monitored up to 30 days after seeding.

### **3.2 Isolation of microbial flora from SMS**

Sample of SMS of *Pleurotus eous* was mechanically stirred with distilled water. The SMS suspension was clarified by centrifugation, serially diluted (10−2 to 10−7) with sterile water and inoculated on potato dextrose agar medium (PDA) and nutrient agar (NA) medium. All the strains observed growing on plates were arbitrarily selected, transferred and maintained in new PDA or NA plates for further use.

A representative sample colony of each visually differentiable bacterium was selected using a sterile inoculating loop. Each colony was transferred by streaking an inoculating loop in parallel lines over four quadrants of NA plate. The plates were incubated at 37o c for twenty-four hour. The isolated colonies were used for initial observations about the shape, color, size and other visual properties of each isolate were recorded. The bacterial strains isolated from the SMS of *Pleurotus eous* were characterized and identified by using the standard procedures.

#### **3.3 Antifungal activity of bacteria isolated from SMS in dual culture plate assay**

The fungal pathogens viz., *Fusarium sp., Alternaria sp., Phytophthora sp.*, and *Aspergillus sp*., were obtained from Biotechnology lab at Post Graduate and Research Department of Botany, Thiagarajar College, Madurai.

Antifungal activity of bacteria isolated from SMS of *Pleurotus eous* mushroom was evaluated in a dual plate assay against four fungal pathogens of plants viz., *Fusarium sp.*, *Alternaria sp.*, *Phytophthora sp.* and *Aspergillus sp.* The bacteria and fungi were cultured in opposing fashion on PDA plates. Bacterial isolate broth (100 μl) of approximately 108 cells/ml was inoculated on PDA plates using spread plate technique. Mycelial agar plugs of one day old culture of the test fungi and the bacterial isolate were inoculated opposite to each other. All the plates were maintained at room temperature and observed for the appearance of zone of inhibition surrounding the bacterial colony where the fungal mycelium failed to grow.