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Soil pH and Microbial Properties as Affected by Integrated Use of Biochar, Poultry Manure and Urea



Otobong B. Iren and Victoria F. Ediene
 
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ABSTRACT

Background and Objective: The use of biochar, a charred organic material, is becoming a sustainable technology that leads to the improvement of highly weathered tropical soils. A study was conducted at the Teaching and Research Farm of the University of Calabar to compare the effects of integrated use of Biochar (B), Poultry Manure (PM) and urea on the pH and microbial properties of soil-grown with Amaranthus cruentus. Materials and Methods: A total of 15 treatments consisting of a sole and combined use of biochar, poultry manure, urea and control were fitted on a randomized complete block design with 3 replications. The organic amendments were applied 2 weeks before sowing while urea was applied 2 weeks after sowing. Composite soil samples (0-15 cm) were taken before and after the experiment for pH and microbial analysis. Results: Soil pH was raised from a strongly acidic level to values ranging from 5.73-6.8 (moderately acid-slightly acid, respectively) pH level after the experiment with the highest obtained in Full B+½PM amended soil. The fungal population was maximum in PM (sole and combinations) (18×103-20×103 CFU g–1) and minimum in sole biochar and control (×10×103 CFU g–1). The bacterial population exhibited a similar trend with the maximum value in PM (sole and combinations) (98×106-148×106 CFU g–1) and minimum in control (×36×106 CFU g–1). Conclusion: This suggests that biochar addition alone did not cause any apparent alteration to the microbial population and diversity compared to its complementary use.

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Otobong B. Iren and Victoria F. Ediene, 2021. Soil pH and Microbial Properties as Affected by Integrated Use of Biochar, Poultry Manure and Urea. Pakistan Journal of Biological Sciences, 24: 90-98.

DOI: 10.3923/pjbs.2021.90.98

URL: https://scialert.net/abstract/?doi=pjbs.2021.90.98
 
Copyright: © 2021. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Biochar is a charred organic material, in the form of charcoal, produced from feedstock to be used in amending degraded soils, sequester soil carbon content and improve soil fertility1. It consists of a fast degradable portion and a recalcitrant portion which is very resistant to biological and chemical oxidation and can persist within the soil system for centuries to millennia to completely mineralize2. Its degradation can take years3 thus resulting in long term carbon (C) sequestration4,5 improving soil quality and health6,7 thereby increasing crop yields8. Biochar is generally of alkaline pH and may alter soil pH in a favorable direction for most crops. The ash content of biochar is primarily responsible for the modification of the soil’s pH. Biochar additions in soils have been reported to increase the pH of acidic soils owing to its alkaline nature9, enhance soil fertility and mitigate climate change by reducing soil N2O emissions10.

Positive effects of biochar on biological communities within soils have been reported; enhanced biological N fixation (rhizobia)11; improved colonization of mycorrhizal fungi. Earthworms showed a preference for biochar amended soils12. Rondon et al.11 found out that increasing biochar amendments in the soil can increase the proportion of N derived from fixation by Phaseolus vulgaris (common green bean) and this increased yields. Biochar amendment is reported to change soil biological community composition and its abundance13 and enhance systemic resistance to pathogens and diseases. However, there is little or no research on the effect of biochar fortification with poultry manure and urea fertilizer on soil microbial communities.

The study was designed to assess the effect of wood biochar fortified with poultry manure and urea fertilizer on soil microbial properties and pH of soil-grown with Amaranthus in acidic soil in Calabar.

MATERIALS AND METHODS

Description of the study area: The experiment was conducted at the Teaching and Research Farm of the University of Calabar, Nigeria. The University of Calabar is situated between latitudes 05°32” and 4°27” N and longitudes 07°15” and 90°28”E. The site is in a degraded rainforest vegetation zone of Nigeria, having a bimodal rainfall pattern with a long rainy season from March-July and a short rainy season from September to the first two weeks of November after a short dry spell in August otherwise called ‘August break’. The mean minimum temperature varies from 21-24°C and the mean maximum temperature from 27-30°C. The mean relative humidity varies between 60-90%. The soil of the experimental area is classified as Ultisol based on the USDA system of classification14. The soils are very strongly acidic and generally low in nutrients. This research project was conducted from May-July, 2018.

Collection and preparation of research materials: Biochar made from wood feedstock was milled using a mechanical blender and sieved with a 4 mm size plastic sieve to obtain its smooth fine powder. Amaranthus seeds and urea were obtained from Agricultural Development Project (ADP) office in Calabar, Cross River State while poultry manure was obtained from the University of Calabar Animal Farm.

Experimental design and treatments: A total of 15 treatments were fitted into a randomized complete block design and replicated three times to give a total of 45 experimental units. The treatments consisted of sole use of biochar, urea and poultry manure and their various combinations as shown in Table 1. Sole application of urea which was regarded as a full dose for Amaranthus according to Iren et al.15 was 60 kg N ha–1 while poultry manure and biochar were singly applied at 20 t ha–1.

Field studies: The experimental site was manually cleared, stumped and marked out with pegs. Flatbeds measuring 1×1 m were made manually using a spade. An alley of 1.5 m was left between blocks and 0.6 m between plots. Biochar and poultry manure was applied to specified plots using broadcast with incorporation method and left for two weeks before sowing Amaranthus seeds to allow mineralization to take place. Urea fertilizer was applied to specified plots two weeks after planting using the band placement method. Banding of the urea fertilizer was about 3.75 cm away from the plant on one side of the seed row and about 5 cm deep15.

Amaranthus seeds were mixed with dried river sand before sowing to ensure the seeds were not planted too close together for proper management of the seed rate desired. The mixture was 70% sand and 30% Amaranthus seeds15. These were evenly distributed directly on drills at a distance of 10 cm between each row. The seedlings were later thinned to one plant per stand a few days after emergence at a spacing of 10 cm between plants. Therefore, the planting distance was 10×10 cm giving a plant population of 100 plants per bed equivalent to 1,000,000 plants per hectare (10,000 m2). The plots were kept weed-free throughout the crop growing period by hand pulling because of the closeness of the plants.

Table 1:Treatment combinations

Soil sampling and processing: Composite soil samples (0-15 cm) were taken before the experiment while at the end of the experiment; soil samples were taken per plot, properly labeled, placed in ice chaste and transported to the laboratory for microbial analysis. Soil samples were also taken before the experiment and at the end of the experiment per plot for soil pH determination. The samples were air-dried, sieved with a 2 mm size sieve and stored for onward analysis.

Laboratory analysis: Samples of biochar and soil were subjected to chemical analysis using standard procedures as outlined by Jones16. Soil pH was determined in 1:2.5 soils: water ratio with a pH meter.

Soil microbial analysis
Media preparation and microbial analysis method
Soil extract agar: One thousand grams each of the experimental soil was weighed into a different conical flask containing 1 liter (1000 mL) of distilled water and stirred vigorously using a stirring rod. The supernatant was filtered and the extract was used to prepare the agar. Fifteen grams of powdered Agar were added to the soil extract boiled to dissolve and sterilized by autoclaving at a temperature of 121°C and 151 bs pressure for 15 min.

Malt extract agar: Malt extract agar was used as a medium for fungi. Then, 26 g of agar was suspended in 1000 mL of distilled and 20 mL of malt extract was added. The mixture was mixed properly and sterilized by autoclaving at a temperature of 121°C in inch for 15 min.

Cultivation and enumeration of bacteria in the experimental plots: One gram of each sample was weighed and thoroughly shaken in 10 mL of distilled water. An aliquot (1.0 mL) was transferred aseptically into the next tube and diluted serially in one-tenth stepwise to 10–6 dilution17. From the dilution of 10–5 of each soil sample, 0.1 mL aliquot was transferred aseptically onto freshly prepared nutrient Agar plates18. The inoculated plates were inverted and incubated at 37°C for 24-48 hrs after which the plates were examined for growth. The discrete colonies which developed were counted and the average counts for triplicate cultures were recorded as total viable bacterial count in the sample.

Isolation, characterization and identification of bacteria from the experimental soils: Pure culture of bacteria was obtained by aseptically streaking representative colonies of different morphological types that appeared on the culture plates on a freshly prepared nutrient agar plate which were incubated at 30°C for 24 hrs. Discrete bacteria colonies that developed were sub-cultured on nutrient agar slopes and incubated at 30°C for 24 hrs. These served as pure stock for subsequent characterization tests. Standard characterization tests were performed in duplicates: Gram staining, catalase test, coagulate test, sugar fermentation test, motility test, methyl red test, Voges Proskauer test, indole test and citrate utilization test. The pure cultures were identified on the basis of their cultural, morphological and physiological characteristics in accordance with the method by Weaver et al.19.

Cultivation and enumeration of fungi: Each sample (1 g) was thoroughly shaken into 10 mL of sterile distilled water and diluted serially. From the dilution of 10–3 of each soil sample, 0.1 mL aliquot was transferred aseptically onto freshly prepared malt agar plate to which 0.2 mL of 0.5% of Ampicillin had been added to inhibit the growth of bacteria18. The inoculated plates were inverted and incubated at 30°C (room temperature) for 5-7 days. The colonies which developed were counted and the average count for the triplicate cultures was recorded as total viable fungi in the sample.

Isolation, characterization and identification of fungi: Pure culture of fungi was obtained by subculturing discrete colonies onto freshly prepared malt agar plates and inoculated at 30°C for 5-7 days. The fungal isolates which developed were further subcultured onto agar slopes and incubated at room temperature for 5-7 days. The isolates which developed were pure cultures which were stored in the refrigerator as stock cultures for subsequent characterization test. The following standard characterization tests were performed in duplicates: Macroscopic examination of fungal growth was carried by observing the colony morphology-Diameter, colour (pigmentation), texture and surface appearance. Microscopic examination was done by needle mount method19,20 and observing sexual and asexual reproductive structures the sporangia, conidial head, arthrospores and the vegetative mycelium. Sugar (glucose, lactose, fructose, sucrose, galactose, maltose and mannose) fermentations were carried out for species identification. The complete identification of fungal isolates was done by comparing the results of their cultural, morphological and biochemical characteristics with those of known taxa19.

RESULTS AND DISCUSSION

The soil used was loamy sand and strongly acidic (pH 5.1). It was low in organic carbon (1.15%), total N (0.08%) and exchangeable K (0.11 cmol kg–1) but high in available P (31.02 mg kg–1) and had exchangeable acidity of 2.00 cmol kg–1. The exchangeable bases (Ca, 2.4 cmol kg–1; Mg, 1.2 cmol kg–1; K, 0.11 cmol kg–1, Na, 0.07 cmol kg–1) were low. Generally, the low soil organic carbon, N and K contents indicates low fertility status thus necessitating the need for additional nutrient supply.

Table 2 shows the nutrient concentration of biochar (1.3% N, 0.05% P, 1.72% Ca, 1.92% K and 1.05% Mg). It had an alkaline pH of 7.8 and an organic carbon content of 35.9%. This confirms the report by Chintala et al.9 who stated that biochar contains an ash component that is usually alkaline and therefore could potentially increase soil pH if added to acidic soil. The organic carbon content of the biochar used falls within that reported by Chan and Xu21 who stated that organic carbon in biochar can vary from 0-91%. The high C:N ratio shows the potential of the biochar to decompose slowly.

The effects sole and complementary use of biochar, poultry manure and urea fertilizer on soil pH are shown on Table 3. Soil pH was raised from a strongly acidic level of 5.1 and 5.4 obtained before experiment and control, respectively to values ranging from 5.733 (moderately acid)-6.8 (slightly acid) pH level after the experiment. The highest pH value was obtained in the soil amended with Full B+½PM (6.8), followed by ½B+Full PM (6.7) and ¼B+¾PM (6.6). This means that biochar when combined with poultry manure (organic manure) has a more positive effect on the pH level of the soil than when combined with urea (inorganic fertilizer). This confirms the assertion made by Chintala et al.9 who reported that biochar contains an ash component that is usually alkaline and could potentially increase soil pH if added to acidic soils. Therefore, combining biochar with poultry manure is of the added advantage as poultry manure contains basic nutrients such as calcium and magnesium which displace hydrogen and aluminum ions that cause acidity. This agrees with the result obtained by Iren et al.15 who then attributed the ability of organic manures to increase soil pH to the presence of these basic cations.

The soil pH values obtained in the different treatments differed from each other with a Standard Deviation (SD) of ±0.394 and a coefficient of variability (CV) of 6.279% showing the level of variability among treatments.

Mean population count: The microbial properties of the soils are discussed to Table 4-5 and Fig. 1a-b. The probable bacterial isolates in the rhizosphere soils of the control and sole biochar plots include Arthrobacter spp., Agromyces sp., Bacillus spp., Klebsiella spp., Micrococcus spp., Pseudomonas spp. and Nocardia spp. while the fungal isolates were Aspergillus niger, Nigrosopra spp., Penicillium spp., Pullularia spp. and Rhizopus spp., while the bacterial isolates for sole biochar plots were Arthrobacter spp., Agromyces sp., Bacillus spp., Klebsiella spp., Micrococcus spp. and Pseudomonas spp., while the fungi isolates were Aspergillus spp., Cladosporium spp., Nigrosopra spp., Pullularia spp., Penicillium spp. and Rhizopus spp.

Table 2:Chemical composition of the amendments used

Table 3:
Effect of sole and complementary use of biochar, poultry manure and urea on soil pH
SD: Standard deviation, CV: Coefficient of variability

Interestingly, the sole poultry liter and its combinations had more diverse species with the bacterial recording: Agromyces sp., Streptococcus spp., Salmonella spp., Enterococci spp., Escherichia coli, Bacillus cereus, Staphylococcus spp., Campylobacter spp., Pseudomonas spp. and Micrococcus spp. while fungi species included: Trichoderma spp., Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, Aspergillus terreus, Alternaria alternate, Nectria spp., Actinomycete spp. Bacillus spp. was the most common occurring bacteria across the various treatments while Aspergillus spp. was the most common fungi specie. The ½ Biochar+½ Poultry +½ Urea combinations yielded much more communities of bacteria and fungi (Table 4-5). The bacterial isolates for the sole urea plots include Arthrobacter spp., Agromyces sp., Bacillus spp., Klebsiella spp., Micrococcus spp. and Pseudomonas spp.

The bacterial and fungal counts across the different treated soils varied from the control and sole biochar treatments (Fig. 1a-b). The sole biochar treatment yielded a slight bacterial (×37×106) population increase from the control (×36×106) but showed the least fungal (×10×103) counts and diversity amongst the treatments. There was a less drastic change in the bacterial population and diversity in response to sole biochar application than when compared with the sole urea (×80×106) and poultry manure (×148×106). The combinations of urea and biochar gave higher microbial counts and diversity than biochar alone (Table 4-5). The combinations of ½ biochar+½ urea, ¾ biochar+¼ urea, ¼ biochar+¾ urea, full biochar+½ urea and ½ biochar+full urea, gave mean counts of 78×106, 70×106, 70×106, 79×106 and 80×106, respectively.

Fig. 1(a-b):
Comparison of rhizosphere, (a) Bacterial and (b) Fungal communities in biochar fortified with poultry manure and urea treated soils

Amongst the various treatment combinations, biochar with poultry manure yielded the highest microbial (bacteria and fungi) mean counts and diversity (Table 4) with ¾ biochar+¼ poultry manure and ¼ biochar+¾ poultry manure having a mean of 98×106. This was closely followed by the ½ biochar+½ poultry manure+½ urea treatments with a mean of 100×106 and the combinations of ½ biochar+full poultry manure with a mean count of 118×106. The non-alteration of the bacterial composition may be attributed to the prevailing environmental factor (high rainfall) in the study area which can influence microbial activities. It could also be a result of the application rate as biochar applied in high quantities is reported to cause a decrease in microbial activity as seen in incubation studies done by Baldock and Smernik22.

Table 4:
Probable bacterial Isolates and their percentage occurrence in biochar fortified with poultry manure and urea treated soils

The low activity of microbes in the sole biochar plots could also arise owing to the feedstock and the recalcitrant nature of the biochar. Although it is chronicled in the literature that biochar can be utilized by microorganisms as a carbon source, evidence still exist that biochar shows great resistance to decomposition and utilization by many microorganisms. According to Smith et al.3 the degradation can take millennia. In other words, only a small fraction of biochar is easily available to microbes. Its recalcitrant nature proves to be the most limiting factor regarding the influence it has on chemical and biological processes within the soil system23. This is the main reason why biochar serves mainly as a tool to sequester carbon within the soil system4-6,24.

The different bacteria and fungi species observed for the different treatment combinations are dependent on the degradability of the substance, the stage of decomposition of the treatment materials and the availability of substrate for utilization by the microorganisms. The less drastic change observed in microbial population and diversity in the rhizosphere of sole biochar applied treatment and the appreciable counts and diversity in the biochar/urea and biochar/poultry manure rhizospheres is an indication that the activities of these organisms are boosted by the urea and poultry materials. The percentage occurrence of bacteria (11.40%) and fungi (9.7%) was highest in poultry manure alone treated soils (Table 4-5).

CONCLUSION

The positive influence of biochar applied either alone or in combination with urea or poultry manure show the potentials of biochar in ameliorating acidic soils. However, the combination of biochar with poultry manure is of the added advantage as poultry manure contains basic nutrients such as calcium and magnesium which displaces hydrogen and aluminum ions that cause acidity thus creating a conducive environment for microbes to thrive. Microbial population and diversity were highest in the combined treatments of either biochar with urea or poultry manure compared to biochar alone. The results, therefore, suggest that biochar addition alone did not cause any apparent alteration to the microbial population and diversity compared to its complementary use.

SIGNIFICANCE STATEMENT

This study discovers the usefulness of using biochar either solely or in combination with either organic or inorganic source of nutrient in ameliorating acidic soils. This study will help the researcher to uncover the critical role of complementary use of biochar with other source of nutrients in creating a conducive environment for microbes to thrive that many researchers were not able to explore. Thus for increase in microbial population and diversity in soils to be attained, sole use of biochar should be minimized and other possible combinations may be arrived at.

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