Abstract

The aims of this study were to characterized the bacteria isolated from the soils of Likouala peat bog area in Congo-Brazzaville. Three (03) composite samples were made from a total of 9 soil samples collected. 1g of soil from each composite sample was cultured on PCA, Mossel and EMB media for counting the total flora. Isolation on Mossel medium and identification of isolates was based on cultural, morphological and biochemical characters by conventional techniques. The method of Petri dishes containing the substrate relating to the search for proteolytic and amylolytic activities was used. The results showed total bacterial loads of 9.50x10⁴; 7.31x10⁴ and 6.39x10⁴ CFU/g on PCA medium; 5.81x10⁴; 5.64x10⁴ and 8.56x10³ CFU/g on Mossel medium versus 2.77x10² CFU/g; 5.1x10² and 1.16x10² CFU/g on EMB medium respectively for samples 1, 2 and 3. The respective total anaerobic mesophilic flora of 3.08x10⁴; 2.93x10⁴ and 3.03x10³ CFU/g on PCA medium; 2.48x10³; 4.83x10³ and 3.32x10³ CFU/g on Mossel medium for samples 1, 2 and 3. The optical densities of the aerobic isolates were between 0.634 and 1 and the enzyme production showed diameters of translucent halos between 1 to 3.2 cm for the proteolytic enzymes and between 1.2 to 2.7 cm for the amylolytic enzymes. The evolution of enzyme production is a function of time and of the isolate. The surface part of the soils of the Likouala peat bog area is rich in aerobic and anaerobic bacteria.

1. Introduction

Peat bogs are unique natural wet ecosystems accumulating an organic material called peat. They cover 3% of the earth's surface, storing a third of soil carbon, the transformation of which could, through positive feedback, aggravate the extent of climate change ¹. Their functioning is likely to be impacted by the effects of change affecting the ecosystem and the environment. The peatification process results from an imbalance between the primary production of organic matter by plants via photosynthesis, its decomposition and its mineralization by microorganisms ². In Congo Brazzaville, they are present in the departments of Likouala, Sangha, Cuvette and part of the Plateaux. The Congo Basin peat bog has an average thickness of 3 m, and concentrates 30 giga tonnes of CO₂, or about the equivalent of at least 15 years of CO₂ emissions from the United States. Congo's moist peat forests store almost 30% of all soil organic carbon found in tropical peatlands (about 30 billion tonnes of carbon) and, 5% of that estimated in peatlands worldwide ^{1, 3}. The department of Likouala is made up of continental and artificial wetlands, an immense expanse of swampy forests with floodplain savannahs, floating meadows along the rivers as well as wooded peat bogs. Several studies carried out on the role of peat bogs in the carbon cycle in the face of global warming show that a rise in temperature leads to a significant increase in microbial activity through intense decomposition of organic matter and therefore a significant release of CO₂ by respiration of microorganisms. It also leads to significant carbon fixation by photosynthesis ⁴. Microflora studies have revealed the presence of large quantities of molds in the peat with concentrations of molds close to 107 per gram of dry peat and up to 23 species of yeasts belonging to the genera Trichosporon, Candida, Rhodotorula and others ⁵ and other species of mycobacteria, pathogens such as M. fortuitum ⁶. Although there is growing interest in the study of microbial diversity in peatlands, the processes maintaining this diversity and its role in the functioning and stability of peatlands remain to date very little explored. This deficit is even greater if we consider the interaction between the effect of microbial activities and that of climate change ⁷. Indeed, despite current knowledge on carbon storage, there are still considerable deficits in the microbiology of these ecosystems. It is with this in mind that we have proposed in this work to characterize the bacteria of the Likouala peat bog area.

2. Materials and Methods

9 soil samples were taken by coring in the Likouala peat bog area in the localities of: Bondoki, Bondzale, Center, Ekolongouma, Ekondzo, Itanga, Makodi, Mbala and Moungouma ⁸. The samples were transferred in sterile plastic bags in a cooler, then transported to the Cellular and Molecular Biology Laboratory of the Faculty of Science and Technology (FST) and stored at 4°C. Of the total of 9 samples, three composite samples were made up for bacteriological analysis.

1g of soil from each composite sample was taken using a precision balance in a first test tube containing 9 ml of sterile physiological water. This tube represents the stock solution of the sample from which decimal dilutions were made with PCA, Mossel and EMB media ^{8, 9}.

Two techniques were used: on the surface of 100μl of each dilution were carried out and deposited in the centers of the Petri dishes containing previously poured and solidified media, then the dishes were incubated in an oven for 24 hours at 37°C, in function of the culture time in the oven ^{10, 11}. And en masse, 1 ml of the solution-dilution in the Petri dishes, the incubated dishes so returned to the oven for 24 hours at 37°C at the rate of one dish per dilution.

The technique consists of measuring the number of bacteria by counting only the viable and cultured cells in a culture medium was used. A significant box contains a number of colonies between (15-150) colonies for specific media and between (30-300) for ordinary media. Petri dishes showing colonies in the form of clusters were eliminated ¹². The number of colonies is given after counting by the following relationship allowing the CFU (Colony Forming Unit) to be evaluated ^{11, 13}.

Colonies were purified on Mossel. A colony is picked with a loop or Pasteur pipette using the striation technique to obtain separate colonies. The dishes are incubated in an oven at 37°C. and then observed after 24 hours. Colonies with an identical appearance are considered pure and stored in sterile eppendorfs containing 900 µl of liquid TBS and 100 µl of glycerol, then kept cool at 4°C ¹¹.

The macroscopic observation carried out after the isolation of the strains focused on the description of the colonies, as reported ¹⁴, in particular: the shape, the appearance, the consistency, the color.

Microscopic observation makes it possible to carry out a morphological study of the cells of a bacterial species. The microscopic study focuses in particular on the shape, mobility and arrangement of the cells. It is used to examine fresh colony samples using a 40 objective optical microscope (GX40) ¹⁵.

Catalase is an oxidoreductase that catalyzes the dismutation of hydrogen peroxide into water and oxygen. The test consists of depositing a drop of hydrogen peroxide diluted to one tenth on the glass slide and adding, using a Pasteur pipette, an isolated colony developed on the agar. If there is effervescence or the presence of O2 bubbles, the activity of the enzyme is catalase positive (Catalase+), the opposite indicates that the bacterium is catalase negative (catalase-) ¹⁶.

Gram staining is used to distinguish and classify bacteria into two large groups: Gram (+) are alcohol-resistant and Gram (-) are alcohol-sensitive and also have an outer membrane which takes on the color of safranin after washing. by alcohol).

In order to determine the involvement of isolates in the formation of peat soils, certain enzymes are sought.

The preserved isolate is subcultured on Mossel, incubated at 37°C then the colony is inoculated in the Erlenmeyer flask containing 20 ml of liquid TSB medium then incubated at 37°C to carry out an overnight culture. Then, 4ml are taken, including 3ml to measure the optical density (O.D) which is an expression of bacterial growth using a spectrophometer calibrated at 600nm and 1ml to assess the enzymatic activity.

1 g of agarose powder is mixed with 1 ml of PBS in 100 ml of distilled water concentrated to 1N. The whole is brought to a boil until the agarose has completely dissolved, left to cool to 55-60°C. Then, 10 ml of skimmed milk were added, homogenize the mixture and pour into Petri dishes; after solidification, the wells are made in the gel and 100 µl of the bacterial suspension are placed in each well, followed by incubation of the Petri dish in an oven at 37°C for 12 hours. The observation of a clear translucent zone (halo) indicates that the isolate produces a proteolytic enzyme and the diameter of each translucent halo is measured ^{11, 17, 18}.

1g of starch and 1.5g of agar are dissolved in 100ml of distilled water and sterilized in an autoclave at 121°C for 15 minutes; then the Petri dishes are poured and the wells are made in the gel then 100 μl of the bacterial suspension after overnight culture are placed in each well and incubated at 37°C. for 48 hours. The revelation is made with the lugol; observation of a translucent zone indicates that the strain produces an amylase enzyme and the diameter of each translucent halo is measured ^{11, 19}.

The results were represented, illustrated and statistically analyzed using Microsoft Excel 2016 software.

3. Results and Discussion

Figure 3 illustrates colonies observed on PCA medium. We note the presence of different shapes colonies: whitish in color, circular, irregular and filamentous.

Figure 4 shows the isolated colonies on Mossel : there are three distinct and visible types of colonies; yellow, pink and orange in color, circular, irregular and rhizoid in shape.

Figure 5 represents the colonies isolated on the EMB medium; two types of colonies are isolated and visible. The presence of colonies of pink and black color of circular shape.

Table 1 below represents the bacterial loads expressed in CFU of the total aerobic mesophilic flora of each sample on the PCA, Mossel and EMB media. On the PCA agar, the total flora of sample 1 is 9.50x10⁴ CFU/g; for sample 2 the total flora is 7.31x10⁴ CFU/g and the sample is 6.39x10⁴ CFU/g. On Mossel, sample 1 has a flora of 5.81x10⁴ CFU/g, sample 2 has a flora of 5.64x10⁴ CFU/g and the flora of sample 3 is 8.56x10³ CFU/g. Finally, on the EMB medium, sample 2 has a total flora of 2.77x10² CFU/g and the flora of sample 3 is 1.16x10² CFU/g.

Figure 6 illustrates the colonies isolated on PCA after mass seeding. A few whitish colonies of circular and filamentous shape were obtained after mass inoculation and incubation at 37°C.

Figure 7 shows the isolated colonies on Mossel after mass seeding. Note the presence of colonies of yellow and pink color, circular and irregular in shape.

Table 2 presents the results of the count expressed in CFU of the total anaerobic mesophilic flora of each sample obtained on the PCA and Mossel media. The rates of total flora depend on the sample. On the PCA, sample 1 has a loading rate of 3.08x10⁴ CFU/g, for sample 2 it is 2.93x10⁴ CFU/g and sample 3 has a loading rate of 3.03x10³ CFU/g. On Mossel, sample 1 has a loading rate of 2.48x10³ CFU/g, sample 2 has a load of 4.83x10³ CFU/g and sample 3 the load is 3.32x10³ CFU/g.

For purification, only aerobic bacteria were used. The different morphotypes obtained after purification of the colonies on Mossel are represented in Figure 8. A total of 51 colonies are isolated and stored in an eppendorf tube containing 900 μl of TSB and 100 μl of glycerol at 4°C. for enzymatic activities.

Figure 9 presents the results of the number of isolates from samples taken from soils in the Likouala peat bog area. A total of 51 isolates were isolated and subsequently analyzed. The distribution of results by sample is as follows: 17 isolates or 33% for sample 1; 15 isolates or 30% for sample 2 and 19 isolates or 33% for sample 3.

Figure 10 shows cell shape after fresh examination with the GX40 objective. The results show that all 51 isolates are catalases positive, aerobic, moderately motile and immobile. These cells are mostly rods, cocci and coccobacilli with a creamy and dry appearance, dominated by the presence of 25 isolates with pink colonies, 24 with yellow colonies and 2 with orange colonies.

Figure 11 shows bacterial cells after Gram staining. Of the 51 isolates; 46 isolates are Gram positive and 5 isolates Gram negative.

On sample 1, a total of 17 isolates were preserved: 9 isolates with pink colonies, 7 isolates with yellow colonies and 1 with orange colonies. Among these isolates; 11 Bacilli, 3 Cocci, 2 coccobacilli and 1 fusiform. All these bacteria are aerobic, moderately motile and Gram positive. These characteristics are shown in Table 3.

On sample 2, a total of 15 isolates were isolated: 8 isolates with pink colonies, 7 isolates with yellow colonies and 1 isolate with orange colonies. We have: 13 bacilli and 2 cocci. These bacteria are aerobic and 12 Gram-positive isolates and 3 Gram-negative isolates. These macroscopic and microscopic characteristics of the isolates are shown in Table 4.

On sample 3, a total of 18 isolates were isolated: 8 isolates with pink colonies, 10 isolates with yellow colonies. 10 bacillus, 7 cocci, 1 coccobacillus and 1 fusiform. These bacteria are aerobic and 16 Gram-positive isolates and 2 Gram-negative isolates. The characteristics of isolates from sample 3 are shown in Table 4.

Table 5 represents the optical density (OD) values which expresses the bacterial growth of some isolates after 24 h of culture on the TSB liquid medium. It varies in different ways. The O.D. measurement was carried out on 22 isolates and the values are between 0.634 and 1. The value 1 represents the largest O.D. value or the cutoff value of the isolates. The different values of the optical density are the different Figure 13 and Figure 15.

Figure 12 represents the diameters of the translucent halos used as an indicator of proteolytic enzyme production. Diameters vary from 1 to 3.8 cm.

Figure 13 shows the variation in optical density and protease production of each isolate. From 22 isolates tested, 18 isolates produce proteases. The representative value is 3.8 cm. The highest diameter is attributed to the RE 2 isolate with a diameter of 3.8 cm while the lowest diameters are observed at the level of the RE 21 and JE 22 isolates at a height of 1 cm and the RE 8, JE 10, JE 42 and JE 43 showed an absence of proteolytic enzyme production.

Figure 14 highlights the halos used as an indicator of amylolytic enzyme production. On the whole the values of the diameters vary from 1.3 to 3.2cm.

Figure 15 below illustrates the variation in optical density and amylolytic enzyme production of each isolate. From 22 isolates tested, 16 isolates produced enzymes capable of degrading starch. 3.2 cm is the representative value and is attributed to isolate RE 3 while isolate RE 4 has the smallest diameter: 1.3 cm. However, with isolates RE 6, RE 7, RE 8, JE 10, RE 2 and JE 22 there is no enzyme production.

Optimization of growth and enzyme production as a function of time is carried out using isolates RE 20, RE 21 and RE 24 in the growth and enzyme production figures.

Figure 16 represents the growth kinetics in three Mossel isolates RE 20, RE 21 and RE 24 as a function of time. The variation of the curves obtained presents the same pace, the same phases with different amplitudes at the level of the 3 isolates. There is an exponential growth phase, a slowing phase, a stationary phase and finally a declining phase. At the level of isolates RE 20, RE 21 and RE 24, from t0 to t1 (0 to 24 hours), the increase in growth is progressive and proportional to time; which corresponds to the exponential phase of growth. From t1 to t2 (24 to 48 hours) this is the slowing down phase. From t2 to t4 (48 to 96 hours), despite the increase in time, a stabilization of growth is observed, thus corresponding to the stationary phase. Beyond 96 hours, the growth (D.O) becomes inversely proportional to the time, it is the phase of decline.

Figure 17 represents the results of the study of the production of amylase and proteases. The production of amylase is variable depending on the time and the isolate. At RE 21, from t0 to t2 (0 to 48 hours), production is average and reaches its optimum at t2 (48 hours). Beyond t3, it becomes average and vanishes at t5. In isolate RE 21, low amylase production is observed from t0 to t1 (0 to 24 hours). It becomes maximum and beyond t3 and inversely proportional to the increase in time and so long is canceled at t5. At RE 24, from t0 to t2 (0 to 48 hours), the production of amylase is zero; it reaches its optimum at t3 (72 hours). Beyond t3, it decreases and vanishes at t5 (Figure 17a). Figure 17b shows the kinetics of proteolytic enzyme production. It is noted that, at RE20 and RE24, from t0 to t1 the production is average, this production reaches its optimum at t3. Beyond t3, it slows down and tends to vanish t5 after. Whereas, in RE21, from t0 to t2 (24 to 48 hours), there is no absence of enzyme production; from t2 to t3 (48 to 72 hours) production becomes average and reaches its optimum at t4 (96 hours). Beyond t4, it decreases and vanishes at t5 (120 hours).

Figure 18 shows the evolution of growth and enzyme production (amylase) as a function of time (120 h). The evolution depends on the isolate; optimal production of amylase is observed with isolate RE 20 during the exponential phase of growth. This production slows down from the stationary phase of growth of the isolate to cancel out in the phase of decline. However, with the RE 21 and RE 24 isolates, during the exponential phase of growth, the production of amylase is average and only reaches its optimum in the stationary phase to cancel out in the decay phase.

Figure 19 represents the development of the growth and production of proteases of isolates RE 20, RE 21 and RE 24 as a function of time. Protease production is time and isolate dependent. A maximum production of protease with RE 20 is observed during the exponential phase of growth, it decreases in the stationary phase and remains stable; against an average production during the exponential phase with RE 21 and RE 24. The production of proteases becomes optimal with isolates RE 21 and RE 24 in the stationary phase of growth and slows down with the phase of decline in growth.

4. Discussion

The count of aerobic bacteria on the PCA medium showed a total flora of 9.50x10⁴ CFU/g in sample 1 compared to samples 2 and 3 which were respectively 7.31x10⁴ CFU/g and 6.36x10⁴ CFU/g. These results are weak compared to those obtained ^{19, 20, 21} which were of the order of 3.4x10⁵ to 5.5x10⁷ CFU /g for areas of natural bogs. However, these values were obtained in Europe and the United States where the vegetation, climatic and geological conditions are different from those of Congo-Brazzaville. These factors would certainly explain this difference because these factors are likely to influence the microflora and therefore the counts of microorganisms ²². In our study, the total anaerobic mesophilic flora depends on the sample. Sample 1 has a bacterial load of 3.08x10⁴ CFU/g, sample 2 has a load of 2.93x10⁴ CFU/g while it is 3.03x10³ CFU/g for sample 3. The comparison of total aerobic and anaerobic mesophilic loads leads to the conclusion that the aerobic flora is greater than the total anaerobic flora. These results can be justified by the fact that the nature of the samples used consisted of superficial peat soils. Our results are consistent ^{19, 23} who states that the microbial population is abundant on the surface of peatlands, because there is more oxygen and where carbon is readily available and decomposable. The counting of aerobic bacteria on Mossel (selective medium without polymyxin) shows a high concentration of bacteria of the Bacillus and Staphylococcus genus, always varying per sample. With sample 1 the flora is 5.81x10⁴ CFU/g, followed by sample 2 at 5.64x10⁴ CFU/g then sample 3 with 8.56x10³ CFU/g. These values still remain below those obtained ²⁴ with a count of the order of 9.3x10⁴ CFU/g of bacteria of the Bacillus genus with soil and pepper samples from Brazzaville (Congo). Several studies have already shown that Bacillus can be isolated from food and soil ^{25, 26}. Compared to counting on EMB medium where the sample loads are 2.77x10² CFU/g; 5.1x10² and 1.16x10² CFU/g respectively for samples 1, 2 and 3. Whereas, with anaerobic bacteria, sample 1 at a rate of 2.48x10³ CFU/g preceded by samples 2 and 3 with respective rates of 4.83x10³ CFU/g and 3.32x10³ CFU/g. The morphological, biochemical and cultural characterization of the isolates made it possible to highlight a diversity of colony forms in bacteria. The isolates purified on the different media showed the presence of various Gram + and Gram- bacteria which were catalase positive in accordance with the media used. Of the 51 isolates obtained, 22 were tested for their enzymatic activity. It appears that, out of the 22 isolates, 4 isolates do not produce proteases, namely RE 8, JE 10, JE 42 and RE 43 against 18 isolates which produce proteases with the best performance attributed to isolate RE 2 with a diameter of 3.8 cm while the smallest diameters are observed at the level of isolates RE 21 and JE 22 with 1 cm in diameter. 6 out of 22 isolates do not produce amylase: RE 6, RE 7, RE 8, JE 10, JE 22 and RE 2 against 16 which produce amylase with 3.2 cm in diameter attributed to the RE isolate 3 while isolate RE 4 has the smallest diameter with 1.3 cm. These results therefore suggest a significant production of proteases compared to amylases in the soils of peatland areas. This is why the follow-up of the evolution of the production of proteases and amylase during growth was carried out according to time with the isolates RE 20, RE 21 and RE 24 in a random manner. From the results of the experiment, it appears that the growth curves in the 3 isolates show the same phases: the exponential phase, the slowing phase, the stationary phase and the declining phase; these curves do not show a latency phase. During the exponential phase of growth, a normal multiplication of bacteria. The growth rate is maximum. The stationary phase results in a balance between the number of bacteria resulting from the multiplication and the number of those which die following the depletion of nutrients, accumulation of toxic waste, environmental conditions becoming unfavorable. During the decline phase, there is more growth, the bacteria die and are decomposed more or less quickly by the enzymes released at the time of their death (web). In the 3 isolates, it is after 48 h (t2) that growth is optimal. The absence of the latency phase is due to the time interval used; DO measurements were taken every 24 hours. Note, however, that the production of enzymes does not depend on bacterial growth, they are two distinct phenomena depending on bacterial metabolism. In fact, in isolates RE 21 and RE 24, the enzyme production reaches its optimum during the stationary phase, whereas with isolate RE 20 the optimum is reached during the exponential phase of growth. These results show that our isolates produce more proteases than amylases with diameters greater than those obtained ^{10, 18}.

5. Conclusion

The objective of this study was to characterize bacteria isolated from the soils of the Likouala peat bog area in Congo-Brazzaville from three (03) composite samples consisting of a total of 9 soil samples collected in the area of Likouala peat bog. It appears from this study that the surface part of the soils of the Likouala peat bog are richer in aerobic than anaerobic bacteria. These bacteria were characterized phenotypically with a dominance of bacteria of the genus Bacillus. 22 isolates showed protease and amylase production capacities over time.

Acknowledgements

We would like to thank our mentor Professor Etienne Nguimbi, Dr Ifo Suspence Averti for her help. We are also grateful to all the teams of the Laboratory of Cellular and Molecular Biology of FST and Laboratory of Remote Sensing and Forest Ecology, ENS-UMNG, Brazzaville.

References