Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Label_Title

Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Label_Title

Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Label_Title

Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Diversity and Biological Activities of Mold Isolated from Bilanko and Ngamakala Peat Bog Soils (Republic of Congo)

Gatsé Elgie Viennechie, Mboukou Kimbatsa Irène Marie Cecile, Ngala-Ngo chanta, Baloki Ngoulou Tarcisse, Morabandza Cyr Jonas*, Nguimbi Etienne

Laboratory of Microbiology, Infectiology and Immunology, ENS-UMNG, B.P 69 Brazzaville

Abstract: The aim of this work was to characterize molds isolated from Bilanko and Ngamakala peat bog soils in the Republic of Congo. After isolation on Sabouraud medium and morphological characterization, monitoring of enzyme production (protease, amylase, lipase and cellulase) and antagonism capacity were carried out using classical techniques. Enumeration showed a higher fungal concentration at point 6 of Ngamakala site with 9.10±2.57.10³ versus 5.40±1.19.10³ CFU/g at point 8 of Bilanko site. 09 fungal genera were identified: Aspergillus, Trichoderma, Penicillium, Beauvaria, Rhizomucor, Mucor, Onychocola, Fasarium, and Scytalidium. The Bilanko site was the most diversified with 07 genera, i.e. 52.94%, compared to 04 genera for Ngamakala, i.e. 48.06%, with a predominance of the genus Aspergillus in the 02 sites. 12 isolates (66.66%) produced amylase, protease and lipase, compared to 18 isolates (100%) for cellulase. The genus Aspergillus (E2L2-Ngamakala) was the most efficient in lipase production with a diameter of 5.4 cm. The genus Aspergillus (E6L3-Ngamakala) was the most invasive isolate on all the other isolates tested with a diameter of 90 mm. These results showed that the peat bog soils at both sites are rich in mold whose biological activities can influence biogeochemical cycles.

Keywords: antagonism, hydrolases, mold, soil, peat bog

Received April 02, 2024; Revised May 03, 2024; Accepted May 10, 2024

Citation: Gatsé Elgie Viennechie, Mboukou Kimbatsa Irène Marie Cecile, Ngala-Ngo chanta, Baloki Ngoulou Tarcisse, Morabandza Cyr Jonas, Nguimbi Etienne, Diversity and Biological Activities of Mold Isolated from Bilanko and Ngamakala Peat Bog Soils (Republic of Congo). American Journal of Microbiological Research, 12 (2). 27-37. January 2024. doi:10.12691/ajmr-12-2-3

1. Introduction

Peat bogs are wetlands that are regularly or constantly saturated with water. Organic matter is only partially decomposed here, due to the low oxygen content, and slowly accumulates to form peat. Wetlands are places where the distinctive soil favors the presence of specialized vegetation that grows in conditions where oxygen is depleted or absent. They are environments with unique terrestrial, hydrological and climatic conditions. In addition to peat bogs, other types of wetlands include marshes, swamps and aquatic grass beds. In these environments, the organic matter produced annually decomposes on site or is transported elsewhere as the water table moves with the seasons [1]. It is the modes of vegetation decomposition and water supply that distinguish peatlands from other types of wetlands. Worldwide, peatlands cover 3% of the world's land area, around 4.632 million km², sequester 30% of the total stock of soil organic carbon and are found in 180 countries [2,3]. They are also home to a wide variety of species. The biological functioning of these ecosystems is strongly linked to microbial activity, which gives these micro-organisms a major role in many functions of peatland soils. Studies have shown that general microbiological activity in peat bogs accelerates, leading to strong decomposition of organic matter and significant release of CO₂, due to the rise in temperature as a result of global warming [4]. These environments, which are all the more precious and seriously threatened, are home to significant biodiversity. These include bacteria, micro-algae, archaea, protists and fungi, the existence of which was questionable at the beginning of the 20th century [5]. Microscopic fungi are heterotrophic micro-organisms, divided into two (02) major groups: yeasts and mold. The latter constitute a heterogeneous group of around 20,000 species belonging to four classes: Zygomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes [6,7]. Many mould species produce a wide variety of secondary metabolites, some of which are of vital importance to humans and used in various fields such as agriculture, biotechnology, health and the environment, while others are used for enzyme production [7]. The Climate change is a threat that could destabilize the entire region. The continued accumulation and preservation of organic matter in a peat bog depends to a large extent on the maintenance of conditions of water saturation, anoxia and reduced activity of microorganisms, particularly molds. Although the study of the diversity of these microorganisms is the object of growing interest, the description of this diversity, the process maintaining this diversity and its role in the functioning and stability of peatlands remains to date very little explored in Congo. It is in this context that our study will focus on the diversity of mold in the soils of the Bilanko and Ngamakala peat bogs, in order to characterize them and assess their impact on the environment.

2. Methods

2.1. Sampling

Peatland soil samples were taken at the Bilanko (Impoh village) and Ngamakala (Lifoula) sites in the Mbé plateau (Pool department). The Mbé plateau is a 6,500 km2 wooded swampy depression occupied by herbaceous savannah, with an altitude of 600 m from Brazzaville to Lefini. Samples were taken randomly from the surface at a depth of 0 to 10 cm. Samples were packaged in sterile vials, labelled and transported to the Cellular and Molecular Biology laboratory of the Faculty of Science and Technology (FST), Université Marien NGOUABI.

Figure 1. Location of sample collection sites

2.2. Enumeration

2.2.1. Preparation of Dilutions

10g of peat soils from each sample were removed and added to 90 mL of sterile physiological water in an Erlenmeyer flask for a stock solution (SM). Using a pipette, 1 mL of the stock solution was withdrawn and transferred to the marked test tube containing 9 mL of physiological fluid, then mixed to obtain a homogeneous 10-1 solution. Each new solution is ten times less concentrated than the previous one [8].

2.2.2. Inoculation and Culture

Using a pipette, 100µL of each dilution (inoculum) was taken and placed in a Petri dish containing Sabouraud medium, which had previously been poured and solidified. This volume was then spread over the entire dish, and each inoculated dish was cultured in an oven at 37°C for 7 days [9,10].

2.2.3. Observing and Counting Colonies

The technique used to count colonies is surface counting. This gives the number of colony-forming units (CFU) sampled. This method only takes into account viable mold colonies that can develop under the growth conditions used [11].

2.3. Purification and Storage

The colonies obtained were purified on Sabouraud medium previously poured and solidified in a Petri dish. Plates were incubated at 37°C for 7 days to obtain distinct, homogeneous colonies. Selected isolates were stored at 4°C in Eppendorf tubes containing 200µL glycerol and 800µL liquid medium (LB) [12].

2.4. Macroscopic and Microscopic Identification

Genus’s identification was based essentially on the determination keys described in the literature, using pure fungal cultures by phenotypic typing based on observation of macroscopic characteristics of the thallus (shape, color, texture) and microscopic characteristics of the filaments (septum, branching, conidia, conidiophore, sporangiospore). By comparing the characteristic data obtained with those of reference works containing mussel identification keys [12,13,14].

2.5. Biological Activity

2.5.1. Culture Conditions

To assess enzyme production, the preserved isolates were transferred to Sabouraud medium, incubated at 37°C for 48 h and then each colony was inoculated into the Erlenmeyer flask containing 20 mL of liquid LB medium, then incubated at 37°C for 48 h. 4mL of each culture was taken, including 2mL to measure optical density (O.D.), an expression of mold growth, using a spectrophotometer calibrated at 600nm, and 1mL to assess enzyme activity [15,16].

2.5.2. Cellulolytic Activity Evaluation

0.5g cellulose and 1.5g agar were weighed, added to 100 mL distilled water, homogenized and sterilized at 121°C for 15 min. Petri dishes were poured, solidified and wells were made into which 50µL of the supernatant, previously centrifuged at 600/min for 10 min, was deposited. The plates were incubated at 37°C for 18 to 48 hours. After incubation, the Petri dishes were flooded with lugol solution for 30 seconds, then rinsed with distilled water. The appearance of the clear zone on the cellulose agar plate indicates that the strain has produced cellulase. The diameters of each halo were measured [16,17,18].

2.5.2. Amylolytic Activity Evaluation

1g starch and 1.5g agar were weighed and added to 100 mL homogenized distilled water, then sterilized at 121°C for 15 min. Petri dishes were poured, solidified, wells were made and 50 µL of the supernatant previously centrifuged at 600/min for 10 min was deposited. The plates were incubated at 370°C for 24 to 48 hours. After incubation, the agar medium was coated with Lugol's solution for 30 seconds, followed by rinsing with distilled water. Starch hydrolysis is indicated by the appearance of the translucent zone around the amylase-producing colony. The diameters of each halo were measured [18,19,20].

2.5.4. Lipolytic Activity Evaluation

1mL olive oil and 1.5 g agar were added to 100 mL distilled water, the mixture was brought to the boil and sterilized in an autoclave at 121°C for 20 min. Once the medium had cooled (45 to 500C), it was poured and solidified. Wells were made and 50µL of the supernatant, previously centrifuged at 600/min for 10min, was deposited. The Petri dishes were incubated at 370C for 24h to 48h. Lipid degradation is characterized by visual observation of the clear, transparent zone on the agar after reaction with diluted Lugol's [19].

2.5.5. Proteolytic Activity Evaluation

Milk agar has been used to demonstrate the presence of proteolytic activity in fungal strains [21]. To demonstrate proteolytic activity, 1.5 g of agarose and 10 mL of skimmed milk were placed in 100 mL of sterile distilled water. Petri dishes were poured, solidified and wells were made into which 50µL of the supernatant, previously centrifuged at 600/min for 10 min, was poured for 10 min. The plates were incubated at 370°C for 12 to 18 hours. Degradation of milk casein is characterized by direct visual observation of the clear, transparent zone on the agar [22,23].

2.5.6. Study of Direct Interactions

Antagonism manifests itself either through competition, hyperparasitism, siderophore production or antibiosis [24]. Antagonistic activity has been studied using the direct confrontation method: the opposing cultures technique. This involves placing two agar pellets, one carrying the pure isolate and the other a pure antagonist, in 90 mm Petri dishes containing Sabouraud medium. These explants come from mushroom cultures. Transplants were made at the same time. Incubation was carried out at 370°C for 7 days. Colony diameters of the two antagonistic fungi were measured every day until the seventh day [25].

3. Results

3.1. Mould Enumeration

Figure 2 shows mold colonies on Sabouraud medium after 7 days of cultivation. Colonies of different sizes, shapes and colors can be observed.

Figure 2. mould colonies on Sabouraud medium after 7 days

Table 1. Mould enumeration for Bilanko and Ngamakala samples

Table 1 shows the fungal loads in the various soil sampling points in the Bilanko and Ngamakala peat bogs. The results show that fungal loads vary according to the site and sampling point. The highest fungal loads were observed at the Ngamakala sampling points compared with those at the Bilanko site, with maximum values of 5.40 ± 1.19.103 CFU/g for the Bilanko site at point 8 and 9.10 ± 2.57.103 CFU/g for the Ngamakala site at point 6 respectively.

Ø Statistical analysis

Figure 3 shows the variation in fungal load at the various sampling points on the Bilanko site. There is a significant difference in the fungal load, and a non-proportional distribution of these loads at the different points. Between points 1 and 2 the difference was significant at p = 0.001; between points 2 and 3 it was significant at p = 0.005; between points 3 and 4 it was significant at p = 0.0001; between points 4 and 5 it was significant at p = 0.002 and between points 7 and 8 it was significant at p = 0.013. On the other hand, a non-significant difference was observed between points 5 and 6, and then between points 6 and 7, with values of p = 0.352 and p = 0.137 respectively.

(a= difference between points 1 and 2; b= difference between points 2 and 3; c = difference between points 3 and 4; d = difference between points 4 and 5; e= difference between points 5 and 6; f = difference between points 6 and 7; g = difference between points 7 and 8)

Figure 4 shows the variation in fungal load at the different sampling points at the Ngamakala site. The results showed a significant variation and a non-proportional distribution of mould loads at the different points of the Ngamakala site. There was a significant difference in mould concentration between points 1 and 2 with p = 0.001; points 2 and 3 with p = 0.0002; points 3 and 4 with p = 0.001; points 4 and 5 with p= 0.022; points 5 and 6 with p= 0.003 and points 6 and 7 with p= 0.001.

(a = difference between points 1 and 2; b = difference between points 2 and 3; c = difference between points 3 and 4; d = difference between points 4 and 5; e= difference between points 5 and 6; f = difference between points 6 and 7).

Figure 3. Variation in mould concentration at the Bilanko site

Figure 4. Variation in mould concentration the Ngamakala site

3.2. Purification of Mold

Figure 5 shows the colonies obtained after purification on Sabouraud medium. Isolation revealed a high diversity of mold.

Figure 5. Pure mold isolates on Sabouraud

3.3. Macroscopic and Microscopic Identification

Figure 6 shows the macroscopic and microscopic characteristics of the mold isolated from the peat bog soils at the Bilanko and Ngamakala sites. A total of 34 isolates were obtained, with 18 (52.94%) from the Bilanko site and 16 (48.06%) from the Ngamakala site. 09 mold genera were identified.

Table 2 shows the number and percentage of genera identified at the Bilanko and Ngamakala sites. Seven (7) genera were counted at the Bilanko site: Aspergillus with 38.88%; Trichoderma and Penicillium with 16.66% each; Mucor with 11.11%; Onychocola, Fasarium and Scytalidium with 5.55% each, compared with four (4) genera at the Ngamakala site: Aspergillus with 56.25%, Rhizomucor with 18.75%, Beauveria with 18.75% and Trichoderma with 6.25%. The genera Aspergillus and Trichoderma were found in both sites with respective percentages of 38.88% and 16.66% for the Bilanko site and 56.25% and 6.25% site.

Figure 6. Macroscopic and microscopic characteristics of mold

Table 2. Number and percentage of fungal genera by site

3.4. Identification of Enzymatic Activities

Of the 34 isolates obtained, 18 isolates were randomly selected for enzymatic activities, including 11 from the Bilanko site and 07 from the Ngamakala site.

3.4.1. Amylolytic Activity

Figure 7 shows the halos indicating starch degradation by the isolates tested. Amylase production is determined to verify the ability of molds to degrade starch. Out of 18 strains tested, 12 showed amylolytic activity.

Figure 7. Demonstration of amylolytic activity by isolate (E2N5 Aspergillus sp1)

Figure 8 shows the amylase production profile of the isolates. In both sites, the ability to degrade amylase is observed in isolates with an average diameter of 1.9 cm for each. At the Bilanko and Ngamakala sites, these were isolates E2N5 (Aspergillus sp1) and E6L3 (Aspergillus sp13) respectively. The lowest mean diameter value for the Bilanko site is 1.1 cm, and 1.8 for the Ngamakala site. Isolates obtained at the Ngamakala site showed more amylolytic activity.

Figure 8. Profile of amylase production by isolates

3.4.2. Cellulolytic Activity

Figure 9 shows halos of cellulose degradation by the isolates tested. Our results showed that all isolates possessed cellulolytic activity, i.e. a percentage of 100%.

Figure 10 shows the variation in cellulase production halos for the different isolates. At the Ngamakala and Bilanko sites, the average diameters obtained range from 1.5 cm for E1N2 (Aspergillus sp2) to 3.8 cm for isolate E4N2 (Aspergillus); and from 1.4 cm for E1L1 (Beauveria sp3) to 1.8 cm for isolate E6L3 (Aspergillus sp13).

Figure 9. Evidence of the cellulolytic activity of the isolate (E2N5 Aspergillus sp1)

Figure 10. Cellulase production profile of different mold isolates

3.4.3. Proteolytic Activity

Figure 11 shows the halos indicating the digestion of casein from skimmed milk. The results obtained demonstrate the ability of molds to degrade proteins perfectly. All 18 isolates used showed proteolytic activity, with halo diameters ranging from 07.3 to 2.1 cm.

Figure 12 shows the protease production profile of different isolates. Average diameters for protease production ranged from 2.1 cm E1N2 (Aspergillus sp2) to 7.2 cm E2L2 (Aspergillus sp15) for both sites.

3.4.4. Lipolytic activity

Figure 13 shows lipase production by the isolates. The ability of molds to produce lipases was tested positive with 17 out of 18 isolates.

Figure 14 shows the lipase production profile of the various isolates tested. The molds found at the Ngamakala site showed impotent lipolytic activity, with mean halo diameter values ranging from 1.1 cm for isolate E6L2 (Rhizomucor sp4) to 5.4 cm for isolate E2L2 (Aspergillus sp15).

Figure 11. Evidence of proteolytic enzyme production by isolates

Figure 12. Protease production profile of different mold isolates

Figure 13. Demonstration of lipase production by isolates

Figure 14. Lipase production profile for different isolates

3.5. Antagonism Test

Biological control of fungal isolates was carried out by antagonism testing, with strains selected randomly according to genus. The effectiveness of antagonistic control depends on the isolate's ability to grow rapidly.

Ø E6L2 (Rhizomucor sp4) and E6L3 (Aspergillus sp11)

Figure 15 illustrates the direct confrontation between E6L2 (Rhizomucor sp4) and E6L3 (Aspergillus sp11). The antagonism test between E6L2 and E6L3 showed that the E6L2 isolate covered the entire dish, while the E6L3 isolate did not. This inhibits mycelial growth, as the E6L3 isolate grown alone occupies a larger surface area than that obtained by the test.

Figure 16 shows variations in the diameters of E6L2 and E6L3 isolates in direct confrontation after 6 days. From day 5, isolate E6L2 occupied almost all of the 90 mm on the entire surface of the dish, whereas isolate E6L3 occupied only 17 mm up to day 6.

Figure 15. Direct comparison between E6L2 (Rhizomucor) and E6L3 (Aspergillus)

Figure 16. Growth trends of E6L2 and E6L3 isolates in direct comparison

Ø E6L3 (Aspergillus sp6) and E2N5 (Aspergillus sp1)

Figure 17 shows the direct comparison between isolates E6L3 and E2N5, all belonging to the Aspergillus genus. The E6L3 isolate occupied the entire surface of the dish, while bypassing the E2N5 isolate, whose growth stopped as soon as it came into contact with E6L3.

Figure 17. Direct confrontation between E6L3 (Aspergillus) and E2N5 (Aspergillus)

Figure 18 shows the growth of E6L3 and E2N5 isolates in direct confrontation. After 4 days of incubation, the E6L3 isolate (Aspergillus sp6) had completely invaded the dish, while the growth of the E2N5 isolate was inhibited and its diameter did not exceed 23.33 mm until day 6.

Figure 18. Growth trend of E6L3 and E2N5 isolates in direct comparison

Ø E1N6 (Aspergillus sp4) and E4N3 (Beauveria sp1)

Figure 19 shows the direct comparison between isolate E1N6 belonging to the genus Aspergillus and isolate E4N3 (genus Beauveria). The fast-growing E1N6 isolate invaded the entire dish without coming into contact with the slow-growing E4N3 isolate.

Figure 19. Direct confrontation between E1N6 (Aspergillus) and E4N3 (Beauveria)

Figure 20 shows the growth of E4N3 and E1N6 isolates in direct confrontation over 6 days. From day 1 to day 3, the growth of both isolates increased proportionally. After 4 days of incubation, isolate E1N6 began to invade the whole box, but did not touch isolate E4N3, whose growth did not exceed 26.33mm. The two mold remained distant from each other until day 6, despite the rapid growth of isolate E1N6.

Figure 20. Growth trend of E4N3 and E1N6 isolates in direct comparison

Ø E1N5 (Aspergillus sp8) and E1L1(Beauveria sp3)

Figure 21 shows the direct comparison between Aspergillus isolate E1N5 and Beauveria isolate E1L1. It can be seen that, while both isolates grew over the days, they remained distant from each other.

Figure 21. Direct comparison between E1N5 (Aspergillus) and E1L1 (Beauveria)

Figure 22. Diameter trends for E1N5 and E1L1 isolates

Figure 22 shows the variation in growth diameters of E1N5 and E1L1 isolates as a function of time (days). Up to the 6th day of incubation, the two molds tested remained distant from each other despite their growth, with the largest values of 51.33mm and 22mm respectively.

Ø E4N4 (Penicelium sp1) and E2N5 (Aspergillus sp1) isolates

Figure 23 shows the direct comparison between isolates E4N4 of the genus Penicelium and E2N5 of the genus Aspergillus. Both isolates develop without inhibiting each other's growth until day 7.

Figure 23. Direct comparison between E4N4 (Penicelium) and E2N5 (Aspergillus)

Figure 24. Growth trend of E4N4 and E1L1 isolates in direct comparison

Ø E3L3 (Trichoderma sp4) and E1L1(Beauveria sp3)

Figure 25 shows the direct comparison between E3L3 and E1L1. The two isolates tested developed independently until they came into contact. Isolate E3L3 grew faster than isolate E1L1.

Figure 25. Direct comparison between E3L3 (Trichoderma) and E1L1 (Beauveria)

Figure 26 shows the evolution of the diameters of E3L3 and E1L1 isolates as a function of time (days). Up to day 6, these isolates grew to a diameter of 80.66mm for E3L3 and 25mm for E1L1.

Figure 26. Evolution of E3L3 and E1L1 isolates in direct comparisons

4. Discussion

The aim of this study was to characterize the mold isolated from the soils of the Bilanko and Ngamakala peat bogs. Soil is a reservoir of various microorganisms, depending on the method used. As highlighted in the previous section, peatlands have a high fungal diversity, with growing interest in peatland functioning and climate change. This is explained by the environmental conditions that govern peat ecosystems.

Mould counts on Sabouraud culture medium showed a varied fungal flora at 2 sampling sites. At the Bilanko site, fungal concentration ranged from (1.20±0.34).10² to (5.40±1.19).10³ CFU/g, compared with (1.30±0.09).10² to (9.10±2.57).10³ CFU/g at the Ngamakala site, depending on the sampling point. These results show that the Ngamakala site has a very abundant population compared with the Bilanko site. Statistical analysis of mold load variations between the various sampling points revealed significant differences between points and between sites. There was also a non-proportional distribution of fungal loads between the different points. This difference could be explained by the variation in physico-chemical parameters from one point to another [30].

After using the phenotypic identification key, 09 genera were identified: Aspergillus; Trichoderma; Penicillium; Beauveria; Rhizomucor; Mucor; Onychocola; Fasarium; Scytalidium. These results are in line with those obtained by Barjac [26] and Waksman and Stevens [27], who assert microbial diversity in the surface layer of peat bogs. In both sites, the genus Aspergillus remains the most represented genus, as shown by Labiod and Chaibras on the isolation of molds from a forest soil in Constantine (Algeria), with a frequency of the genus Aspergillus of 50% [10]. Our results also concur with those of Abdlaziz, where the genus Aspergillus was in the majority with a frequency of 37.5%, grouping together 6 different species belonging to the genus Aspergillus, from arid soil [28].

These fungal genera are present in the majority of soils of all kinds; Alvarez-Rodriguez et al. and Boiron have stated that the genera: Aspergillus, Penicillium, Fusarium, Trichoderma, Mucor, are autochthonous strains, usually isolated from most soils [6,29]. The number and activity of these populations has been shown to vary from region to region, and may be influenced by soil organic matter content, soil texture, pH, moisture, temperature, aeration and other factors [31].

In order to better understand the involvement of molds in peatification, the production of four enzymes was demonstrated: amylases, cellulases, proteases and lipases. The results showed that all 18 isolates tested were capable of producing at least three (03) enzymes. The enzymatic activity of microorganisms is directly influenced by the availability of nutrients in the environment, and is part of the adaptation of these microorganisms to their natural environment. Protease production was tested positive with all isolates. According to [32,33], proteases are the most important enzymes that can be produced by several fungal genera such as Trichoderma, Mucor, Rhizomucor, etc. These results allow us to consider these isolates as producers of exocellular proteases [34]. Cellulose degradation was observed in all isolates tested. This can be explained by the fact that the rate of cellulose decomposition depends on the microorganisms involved and the ecological conditions. These results are in line with work carried out by [35,16], which demonstrated the ability of fungal genera, notably Trichoderma, to produce cellulase. With regard to amylase production, 12 isolates were able to degrade starch. This ability of fungal isolates to degrade starch has already been demonstrated in a study by Tatsinkou and colleagues [19], who showed the ability of certain fungal strains to produce alpha amylase. Lipase production was most significant in isolates from the Ngamakala site, which showed significant lipolytic activity, with mean halot diameters ranging from 5.4 cm for Aspergillus isolate E2L2 to 1 cm for Rhizomucor isolate E6L2.

The results of the antagonist test show that there are different antagonistic effects with the isolates tested. Studies by Laclere [36]; Vannacci and Herman, [37] have shown that many fungi produce compounds with properties that can interfere with the growth and activity of other fungi. Direct confrontation between isolates E6L3, E1N6, E1N5 and E1N6, all belonging to the genus Aspergillus, and isolates E2N5, E4N3, E1L1 and E6N4 respectively, showed a reduction in mycelial growth in the colonies of the latter. Indeed, the genus Aspergillus is renowned for its ability to produce inhibitory molecules against other fungal strains. Seidl and colleagues [38] have confirmed this by comparing Aspergillus niger with other fungal strains. E6L2 of the genus Rhizomucor. Benmechirah and Lidjici [39], working on the antagonistic activity of molds, have demonstrated the inhibition of Fasarium solani mycelium growth in relation to Aspergillus niger. This inhibition of fungal growth may be due to exponential growth leading to the secretion of harmful extracellular compounds such as xylanases, enzymes that degrade the cell wall. In the case of direct confrontation between isolates E6L2 and E6L3, we can see that isolate E6L2 is growing rapidly and has covered isolate E6L3. It does not allow the latter to grow below the colony at the same time [40].

5. Conclusion

This work phenotypically characterized molds in Bilanko and Ngamakala peat bog soils, their enzymatic production and antagonism. Results showed a richness of molds in both sites with high fungal loads, 09 genera identified: Aspergillus; Trichoderma; Penicillium; Beauvaria; rhizomucor; Mucor; Onychocola; Fasarium and Scytalidium. Good enzyme production was observed with lipase diameters of 5.4 cm with Aspergillus (E2L2-Ngamakala) and strong antagonistic activity with 90 mm diameter with the genus Aspergillus (E6L3-Ngamakala). The Bilanko and Ngamakala peat bogs contain a diversity of molds capable of producing enzymes and inhibitors.

References

[1]   | Payette S., Rochefort L. 2001. Ecologie des tourbières du Québec-Labrador, Saint-Nicolas, les presse de l'université laval, 85P.
[2]   | Leifeld, J., Menichetti, L. 2018. The underappreciated potential of peatlands in global climate change mitigation strategies. Nat. Commun. 9, 1071.

[3]  
Minasny, B., Berglund, Ö., Connolly, J., Hedley, C., de Vries, F., Gimona, A., Kempen, B., Kidd, D., Lilja, H., Malone, B., McBratney, A., Roudier, P., O’Rourke, S., Rudiyanto, Padarian, J., Poggio, L., ten Caten, A., Thompson, D., Tuve, C., Widyatmanti, W. 2019. Digital mapping of peatlands–A critical review. Earth-Sci. Rev. 196, 102870.

[4]   | Richy E. 2021. Rôle de la diversité des microorganismes sur le fonctionnement des tourbieres dans un contexte de changement climatique ; Thèse de doctorat, Université de Pau et des Pays de l'Adour ; 237pp.
[5]   | Dommergues Y. 1997. Contribution of actinorhizal plants to tropical soil productivity and rehabilitation. Soil Biology and Biochemistry, 29(5), P. 931-941. Sustainable agriculture for the Tropics: the role of Biological Nitogen fixation, Rio de Janeiro (BRA), 1995/11/26- 1995/12/01, ISSN 0038-0717.
[6]   | Boiron P. 1996. Organisation et biologie des champignons. Edition Nathan.p:13-69.
[7]   | Bousseboua H. 2005. Eléments de microbiologie. Vol.1, 2nd edition. Campus-Club, Algeria, 304p.
[8]   | Bonnefoy et al. CB. 2002. Microbiologie et qualité dans les industries agroalimentaires. (Figarella, Ed.) Courtry, France: Biosciences et Techniques.
[9]   | Abdelaziz W. 2006. Isolation of antibacterial substance-producing fungi from Saharan soils. Mémoire de Magistère En Microbiologie et Biochimie Appliquées Université Mentouri. Constantine 55P.
[10]   | Labiod F. and Chaibras S. 2015. Isolation, identification and antibacterial activity of molds from a forest soil in Constantine, Master's thesis, Université des Frères Mentouri Constantine, 74pp.
[11]   | IRSST. 2011. Dénombrement des bactéries et des moisissures cultivables des l'air prélevé sur filtre de polycarbonate, IRSST 3eme révision, 8pp, http://www.irsst.qc.ca/fr /methodes.
[12]   | Botton B., Breton A., Fèvre M., Gauthier S., Guy P., Larpent J.P., Reymond P., Sanglier J.J., Vayssier Y., Veau P. 1990. Useful and harmful molds: Industrial importance. 2nd Edition. Masson. Paris, 512p.
[13]   | Malloch, DM. 1997. Moisissures, Isolation, culture, identification. Toronto.
[14]   | Guiraud J. P. 1998. Microbiologie alimentaire. Dunod. Paris.778pp.

[15]  
Nguimbi E., Ahombo G., Moyen R., Ampa R. ; Vouidibio A., Ontsira E. N., Kobawila S. C., Louembe D. 2014. Optimization of Growth, Fibrinolytic Enzyme Production and PCR Amplification of Encoding Fibrinolytic Enzyme Gene in Bacillus amyloliquefaciens Isolated from Ntoba mbodi at Brazzaville. International Journal of Science and Research (IJSR), Volume 3 Issue 11, 2319-7064.

[16]  
Gatse E.V., Mboukou K.I.M.C., Onyankouang I. S., Morabandza C. J., Goma Tchimbakala J. and Nguimbi E. 2023. Monitoring of Cellulase and Biosurfactant Production in Bacteria Isolated from the Likouala Peat Bog Area, (Republic of Congo). International Journal of Environmental Bioremediation & Biodegradation, vol. 11, n°.1.1-8.

[17]   | Fergani et Lakhel. 2015. Activités cellulolytiques de Thrichoderma longibrachiatum cultivée sur son de blé. Mémoire Master: Biotechnologie des mycètes. Canstantine: Université Mentouri, 56.
[18]   | Ngô I., Nguimbi E., Kayath, C., Ampa, R. 2019. Molecular Identification and Phylogenetic classification and Proteolytic capacity of cultivable Bacteria Isolated from soils in Brazzaville, Republic of Congo. Journal of Biochemestry,Microbiology and Biotechnology.
[19]   | Tatsinkou F.B., Taveai F. et Ndjouenkeui R. 2005. Production and partial characterization of a thermostable amylase from ascomycetes yeast strain isolated from starchy soils. African Journal of biotechnology, 4(1), p 14-18.
[20]   | Morabandza C.J., Gatsé E.V., Mboukou Kimbatsa I. M. C., Onyankouag, I.S., Ifo S. A., and Nguimbi E. 2022. Characterization of Isolated Bacteria from Soils in the Likouala Peat Bog Area (Republic of Congo). American Journal of Microbiological Research, 10, 59-70.
[21]   | Clarke P-H. et Steel K.J. 1966. Rapide and simple biochemical tests for bacterial identification. Academic press. London, p 111.
[22]   | Harrigan W.F. et Mccance M.E. 1976. Laboratory methods in food and dairy microbiology. Academic press. London. p 21-277.
[23]   | Benkahoul M., Belmessikh A., Boukhalfa H. et Mechakra-Maza A. 2017. Optimisation à l’aide d’un plan d’expériences de la production d’une protéase fongique sur milieu à base de déchets agro-industriels. Journal Article, sciences et techniques issue N°75.
[24]   | Soufiane B. 1998. Isolement à partir de la rhizosphére des conifers de Bactéries et d’actinomycètes antagonistes aux champignons phytopathogènes. Canada, pp56.
[25]   | Benhamou N. and Chet L. 1996. Parasitism of sclerotia of Sclerotium rolfsii by Trichoderma harzianum ultrastructural and cytochemical aspects of the interaction. Phytopathology 86, p. 405-416.
[26]   | Barjac, H. 1955. Essai d’interprétation bactériologique de sols tourbeux acides. Thèse de doctorat en Sciences Naturelles. Université de Paris (France). 160p.
[27]   | Waksman, S.A. et Stevens, K.R. 1929. Contribution to the chemical composition of peat: V. The rôle of microrganisms in peat formation. Soil Sci. 27: 315-340.

[28]  
Alvarez, Ropdriguez M.L., Lopez-Ocana L., Lopez C., Rodriguez N.E., Martinez M.J., Larriba G & Coque J-J.R. 2002. Cork taint of wines: role of filamentous fungi Isolated from rock in the function of 2,4,6- Trichloroanisol by O methylation of2,4,6 – Trichlorophenol. Applied and Environmental Microbiology. 68 (12): 5860-5869.

[29]   | Peuk A.D. 2000. The chemical composition of xylen sapin Viritis vinifera L.cv. Riesling during vegetative growth on three different francian vineyard soils and as influenced by nitrogen fertilizer.Am. Enol. Viticult. 51:329-339.
[30]   | Smith N. R., Gordon R. E. & Clark F.E. 1952. Aerobic spores-forming bacteria. J. Appl. Bact. 27: 78-99. In thèse de doctorat.
[31]   | Frazier W-C. 1967. Food microbiology. Academic presse. London. p 03-429.
[32]   | Ui-haq I., Mukhtar H., Daudi S., Sikander A., et Quadeer M. A. 2003. Production of proteases by a isolated mould culture under lab condition. Biotechnology 2 (1), p 30-36.
[33]   | Duce R. G. et Thomas S. B. 1959. The microbiological examination of Butter. J. Appl.
[34]   | Dommegues Y. et Mangenot F. 1970. Ecologie microbienne du sol, Masson et Clé, Paris, 783p.
[35]   | Leghlimi H. 2004. Optimisation de la production de la cellulase d’Aspergillus niger ATTCC16404 cultivé sur un milieu à base de lactosérum: étude comparative entre Aspergillus niger ATCC 16404 et Aspergillus niger O.Z isolée localement. Université Mentouri Constantine.
[36]   | Leclère V, Béchet M, Adam A, Guez JS, Wathelet B, Ongena M, Thonart P, Gancel F, Chollet-Imbert M, Jacques P. 2005. Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism's antagonistic and biocontrol activities. Appl Environ Microbiol. Aug ;71(8): 4577-84.
[37]   | Vannacci G. et Harman GE. 1987. Biocontrol of seed-borne Alternaria raphaniand A. brassicicola. Canadian Journal of Microbiology 33: 850–856.
[38]   | Seidl V., Marchetti M., Schandl R., Allmaier G., et C.P. Kubicek. 2006. Epl1, the major secreted protein of Hypocreaatroviridis on glucose, is a member of a strongly conserved protein family comprising plant defense response elicitors. FEBS J. 273: 4346-4359.
[39]   | Benmechirah N, D & Lidjici Y. 2019. Activités hydrolytiques et antagonistes des moisissures isolées depuis le lac d’eau douce «Timerganine ». Université des Frères Mentouri Constantine. Faculté des Sciences de la Nature et de la Vie. 75p.
[40]   | Biljana G., Jugoslav Z. 2011. Trichodermaharzianum as a biocontrol agent against Alternariaalternata on tobacco. Appl. Technol. Innov. 7: 67-76.