CHAPTER FOUR

RESULTS

4.1 Sample Collection and Isolation of Bacillus Species

Six soil samples, including soil from marshy places, animal feces, dry leaf

residues, stored product dust, and other decomposed organic matter, were collected

from various locations. The samples were processed for the isolation of Bacillus

species using sodium acetate-enriched nutrient broth. Heat treatment was applied

to eliminate non-spore-forming bacteria, ensuring selective isolation of Bacillus

species.

4.2 Morphological Characteristics of Isolated Bacillus Species

The isolated Bacillus colonies displayed diverse characteristics, including creamy,

rough edges, and a spread-out growth pattern. Table 4.1 summarizes the colony

morphology and distribution observed from the six samples:

Sample Code Colony Morphology Percentage (%) of Isolates

A (0.12 m) Creamy, slightly raised, rough edges 25%

A (0.25 m) Creamy, opaque, serrated edges 37%

A (NSA) Creamy, spreading colony 38%

B (0.12 m) Dull opaque, flat, smooth edges 36%

B (0.25 m) Creamy, rough edges, glistening 27%

B (NSA) Creamy, flat, spreading, rough edges 37%

4.3 Biochemical Characterization

The isolates were confirmed as Bacillus species through gram staining and a series

of biochemical tests. All isolates were Gram-positive rods and showed positive

results for catalase and oxidase activity, indicating oxidative metabolic pathways.

Table 4.2 presents the biochemical test results for all six isolates:

Isolate Code Catalase Oxidase Spore Test Sugar Fermentation (Glucose)

A1 + + + +

A2 + + + +

B1 + + + +

B2 + + + +

C1 + + + +

C2 + + + +

4.4 Bioassay Against Cockroach

The biocontrol efficacy of Bacillus isolates was tested against cockroaches using

direct contact bioassays. Serial dilutions of Bacillus spores were prepared, and the
mortality rate of cockroaches was observed over a 48-hour period. Higher

concentrations (10⁻¹ to 10⁻²) demonstrated significant mortality compared to

lower dilutions. Table 4.3 presents the mortality percentages recorded from the six

samples:

Dilution Mortality Rate (%) Observation Period (Hours)

10⁻¹ 80% 24

10⁻² 65% 24

10⁻³ 40% 24

4.5 Discussion

The six Bacillus isolates demonstrated significant biocontrol potential against

cockroaches. The observed mortality rates confirm the efficacy of Bacillus isolates,

particularly at higher concentrations, where increased toxin availability led to

higher mortality rates.

This study corroborates the findings that Bacillus species, including Bacillus

thuringiensis, are potent biological control agents due to their production of

insecticidal crystalline proteins. These proteins disrupt the physiological processes

of pests, causing cell lysis and eventual mortality. The results emphasize the

reliability of Bacillus isolates from soil samples as environmentally friendly

alternatives to chemical pesticides.

SUMMARY, CONCLUSION, AND RECOMMENDATIONS

5.1 Summary

This study investigated the biocontrol potential of Bacillus species isolated from

soil samples against cockroaches. Soil samples were collected from six different

locations with varied environmental conditions, such as marshy areas, animal

feces, and decomposed organic materials. These locations were chosen to ensure a

broad range of Bacillus species with potential biocontrol efficacy.

The samples were processed using sodium acetate-enriched nutrient broth to

selectively isolate spore-forming Bacillus species. Heat treatment was applied to

eliminate non-spore-forming bacteria, resulting in the isolation of six distinct

Bacillus strains. These isolates were subjected to morphological and biochemical

characterization to confirm their identity as Bacillus species.

Morphological studies revealed that the Bacillus isolates had distinct colony

structures, including creamy textures, rough edges, and spreading growth patterns.

Biochemical tests, such as catalase, oxidase, and sugar fermentation tests,

confirmed the oxidative and spore-forming capabilities of the isolates, which are

critical for their biocontrol properties.

direct contact bioassays. The bioassays involved serial dilutions of Bacillus spores,

with mortality rates recorded over 48 hours. Higher concentrations of Bacillus

spores showed significant insecticidal effects, causing mortality rates of up to 80%,

while lower concentrations demonstrated reduced efficacy.

The results demonstrated that the insecticidal crystalline proteins produced by

Bacillus species were effective in disrupting the physiological processes of

cockroaches, leading to their mortality. This finding aligns with prior research

highlighting the potential of Bacillus species, particularly Bacillus thuringiensis, as

eco-friendly alternatives to chemical pesticides.

Overall, this study establishes the effectiveness of Bacillus isolates as biological

control agents against cockroaches, reinforcing the importance of using microbial

biopesticides in pest management strategies. This approach provides a safer, more

sustainable alternative to chemical insecticides, with minimal environmental

impact.

5.2 Conclusion
The findings of this study confirm the significant biocontrol potential of Bacillus

species isolated from soil samples against cockroaches. The isolates demonstrated

robust insecticidal activity, particularly at higher concentrations, highlighting their

efficacy as biological control agents.

The morphological and biochemical characterization confirmed the presence of

spore-forming Bacillus species capable of producing insecticidal crystalline

proteins. These proteins disrupt the gut lining of target pests, causing mortality,

and represent a natural, eco-friendly solution for pest management.

This study underscores the importance of exploring soil microbiota as a source of

biocontrol agents. The diversity of Bacillus isolates obtained from various soil

samples indicates the potential for discovering more potent strains with broader

applications in pest control.

The results contribute to the growing body of evidence supporting the use of

microbial pesticides as an alternative to synthetic chemicals. By leveraging the

natural biocontrol properties of Bacillus species, it is possible to achieve effective

pest management while minimizing environmental and health risks.

5.3 Recommendations
1. Future research should explore the genetic diversity of Bacillus species

isolated from various soil environments to identify strains with enhanced

biocontrol efficacy.

2. Industrial-scale production of Bacillus-based biopesticides should be

developed, ensuring high-quality products with consistent insecticidal

activity.

3. Field trials should be conducted to assess the efficacy of Bacillus

biopesticides under real-world conditions, particularly in urban settings

where cockroach infestations are common.

4. Public awareness campaigns should be implemented to educate communities

on the benefits of using microbial pesticides as safe, eco-friendly alternatives

to chemical insecticides.

5. Policies and regulatory frameworks should be established to promote the use

of biological control agents, encouraging their integration into existing pest

management practices.