Indigenous Peat Cellulolytic Bacteria and Its Potential as A Liberica Coffee Growth Promoter

. Among the main microbes in peat are cellulolytic bacteria. The research aimed to select peat cellulolytic bacteria and identified its potential as a plant growth promoter bacteria (PGPB). The cellulolytic bacteria were isolated by serial dilutions and cellulase activity by the carboxymethyl cellulose (CMC) method, species types recognized by the sequencing method and P solubilization and phytohormones productions by Pikovskaya, and the high-performance liquid chromatography method, respectively. Research results found the 1 st identified peat cellulolytic bacteria, Comamonas testosteroni, dissolved

The PGPB presence could reduce some chemical fertilizer roles and the risk of environmental damage due to land cultivation [9].Some cellulolytic bacteria isolated from Indonesian soils had been identified as having potential as the PGPB because of their ability to dissolve P and fix N.They are Bacillus stratosphericus, Bacillus amyloliquefaciens, Bacillus cereus, Pseudomonas pseudoalcaligenes, Pantoea dispersa, and Sinomonas atrocyanea [8].
The Liberica coffee plant is one of three commercial coffees in Indonesia besides Robusta and Arabica.Liberica coffee grows larger than Robusta and Arabica plants, of which the crown diameter is about 3.5 -4 m and a height of about 5 m, and also has sizer cherries and beans and leaf area [10]; [11].The Liberica coffee plantation is more suitable for the peatlands [12]; [13]; [14].However, the fertility of peatland is low because of low nutrient content like available P [14].
In this study, cellulolytic bacteria isolated from peat were followed by identifying bacterial species, testing cellulase activities, the ability to dissolve fixed P and produce phytohormones, and then testing their application effect on available P and organic C of peat and the Liberica coffee seedling growth.So, the research was to select peat cellulolytic bacteria and to identify its potential as a plant growth promoter bacteria.

Peat sample
For microbial isolation, peat was taken on a smallholder plantation of Liberica coffee on peatland in Tanjung Jabung Barat, Province of Jambi, Sumatra.The peat was sampled on the coordinates of -0⁰59'(11"-27") S and 103⁰21'(0"-42") E at a depth of 0-20 cm, mixed, and put into waterproof plastic bags, and stored in a cool box.The peat sample for the greenhouse experiment, approximately 600 kg, was also from the same peatland.The analysis results for several peat chemical properties such as pH (a glass electrode method), organic carbon, and ash [loss on ignition (LOI)], total N (Kjeldahl method), and P 2 O 5 (Bray method) are in Table 1.

Cellulolytic bacterial isolation
The method to isolate the cellulolytic bacteria was the serial dilutions method, started by putting ten grams of peat sample into 90 mL of physiological solution (0.85% NaCl solution) in an Erlenmeyer and rotated for 30 minutes at a speed of 150 rpm by a rotary shaker, and then diluted for a concentration of 10 -2 up to 10 -5 .Next, one mL of microbial cultures from dilutions of 10 -3 , 10 -4 , and 10 -5 were grown in the Nutrient Agar (NA), incubated for three days at room temperature, and then the bacterial colonies formed on the plates were counted.

Cellulase activity
The cellulase activity of bacteria qualitatively was by the CMC method (the mixture of 0.4 g CMC (Merck), 0.5 g MgSO4.7H2O,0.03 g KNO3, 1.0 g K2HPO4, 0.0008 g FeSO4.7H2O,0.08 yeasts, 2 g NaNO3, and 18 g agar), which was all dissolved into a liter of distilled water, into which the pure bacterial colonies were spread then for 24 hours was incubated at room temperature pre-drop it with 0.1% (w / v) Congo red solution.The presence of clear zones around the bacterial colony indicated a cellulolytic enzyme (cellulase activity) by which the most active bacteria was with the higher ratio of clear-zone diameter and bacterial colony diameter of cellulolytic bacteria (AI).The formula to calculate the qualitative cellulolytic index is: The activity index (AI) = Diameter of the clear zone (mm) in CMC Diameter of the bacterial colonies (mm) in CMC (1) Fig. 1 The clear zone around the bacterial colonies by cellulase activity of bacteria in CMC Then the Selected bacteria were grown in media consisting of a mixture of 2.0 g KH 2 PO 4 , 1.4 g (NH4) 2 SO 4 , 0.3 g MgSO 4 .7H 2 O, 0.1 g CaCl 2 , 1.0 g peptone, and 2.0 g agar and incubated for seven days on slant agar at 37 °C.Then, the enzyme solution (supernatant) was separated from the particle of substrate or bacterial cell by centrifugation of bacterial culture at 13,000 rpm and 4⁰ C for 10 min and then kept in a freezer -10⁰ C. Then the quantity cellulase activity measurement was by the DNS (dinitro salicylic acid) method [15].The assay for the cellulase activity of the cellulolytic bacterial colonies was by Duplo analysis.

Cellulolytic bacterial genotypic identification
Identifying bacterial genotypes of some selected cellulolytic bacteria was done by sequencing primer 16sRNA.The process of identifying bacterial genotypes was by the nucleotide base sequencing technique by aligning the sequences with BioEdit 7.7 sequence alignment editor, then compared to the GenBank database using Basic Local Alignment Search Tool (nih.gov)(https://blast.ncbi.nlm.nih.gov/Blast.cgi.)

Phosphate solubilization
The potential of cellulolytic bacteria as a phosphate solubilizer was assayed in vitro using Pikovskaya agar [16]; [17].One ml of bacterial colony isolate was put into Pikovskaya in an Erlenmeyer and shaken at 100 rpm for two weeks.After two weeks, 20 ml of the supernatant was filtered with Whatman paper no. 1 into the centrifugal tube and centrifuged at 1000 rpm for 15 minutes.Then was transferred 5 ml into a test tube, added 0.5 ml of concentrated dye reagent, shaken for several minutes, and incubated for 30 minutes.The standard solution is 1 ml of 1000 mg/kg K 2 HPO 4 diluted to concentrations of 0, 2, 4, 6, 8, and 10 mg/kg, respectively.Add 0.5 ml of P reagent to each standard series, shake for a few minutes, and incubate for 30 minutes.The solution absorbance of each concentration series, and in Pikovskaya media inoculated by bacterial colonies isolates, was measured by a spectrophotometer at a wavelength of 693 nm.The PO 4 of the standard solution series graph was used to calculate the dissolved P of bacterial colony isolates, with the formula: Bacterial isolate PO 4 (mg/kg) = curve mg/kg x fp -PO 4 control concentration (mg/kg) and fp is the dilution factor (if any).

Assay the effect of cellulolytic bacteria enrichment on Liberica coffee seedling growth
It was a pot experiment conducted in a greenhouse (GH).Fresh peat enriched by peat indigenous cellulolytic bacteria, planted with 9-month-old Liberika coffee seedlings, were compared with fresh peat in pots with no bacterial enrichment.Cellulolytic bacterial colonies enrichment (10 8 CFU/mL) was applied as much as 25 mL to 5 kg of fresh peat for ten pots.After bacterial inoculation, mixed peat thoroughly and incubated for a week.There was no addition of chemicals and other manure to the peat.To keep the peat moist, water at a plastic dish at the pot bottom at a level of 5 cm was maintained.Observations were on the Liberika coffee seedling growth, like stem growth (height and diameter) and the number of leaves.A comparison of the variable data was analyzed using the T-test at a 5% significance level.

Cellulolytic bacterial index and activity
By the serial dilutions method, 486 bacteria grew from peat samples and at CMC media, 31 of which formed clear zones.The bacterial colonies with sample codes BT-19, BT-21, and BT-26 had higher cellulase activity indexes than others (Table 2) and the highest cellulose enzymes released by the bacterial colony of sample code BT-21 (Table 3).The presence of cellulolytic bacteria in peat is in line with what was stated by [19], namely cellulolytic bacteria were among the microbes that are adaptive to the aquatic environment like peat, which is rich in carbon as the essential source of microbes' energy [20].Note: BT = Bacteria, *1 Unit (U) = 1 μmol product / minute

Cellulolytic bacterial index and activity
The results of the sequencing method showed the bacterial isolate of BT-19 is similar at 81% to Comamonas testosteroni strain NBRC 14951, while the similarity of the bacterial isolate of BT-21 and BT-26 are 91% and 88% to Delftia lacustris strain 332, respectively (Table 3).The consortia of Comamonas sp are able to degrade lignocellulose [21]; [22].They live in an organic acid environment [23], wetland environments, activated sludge, and sediments in forest soils [24]; [25].
The bacterium Delftia lacustris strain 332 isolated from freshwater, was first identified in Denmark [26].The Delftia sp was easily adaptive to aqueous/wet environments such as freshwater, marine, rhizosphere, soil, and so on [27].

Phosphate solubilization
The analysis result showed that Comamonas testosteroni and Delftia lacustris could dissolve fixed P, of which the isolate of Comamonas testosteroni colonies' ability to dissolve 1.908 µg PO43-/mL.day) is better than Delftia lacustris namely from 1.107 µg PO43-/mL to 1.329 µg PO43-/mL.day(Table 4).The capability of Comamonas testosteroni to dissolve P was also previously reported [28], even though it solubilized potassium and fixes N as well.
Another experiment reported the consortia of bacterial taxa, including Comamonas testosteroni, inoculated into inorganic fertilizers to mobilize soil bound-P increased the productivity of several crops such as Jalapeno and wheat up to twofold [29].The ability of Delftia lacustris to solubilize P was also reported by Agafonova et al. [2] and Tamás et al. [30].

Phytohormones production
The cellulolytic bacterium Comamonas testosteroni a facultative anaerobic bacterium could release the plant growth regulator hormones such as auxins (IAA), gibberellin, zeatin, and kinetin, while Delftia lacustris produced IAA, gibberellin, and zeatin and no kinetin (Table 5).Previously the Comamonas testosteroni was identified in the banana rhizosphere, could produce siderophore [31], and had been used as a good bio-fertilizer [32].In the USA, an experiment found that Comamonas testoteroni could be a bio-fertilizer for flax crops (Linum usitatissimum L), which were growing under salinity stress.Its ability to alleviate the salinity is threatening because it reduced photosynthesis pigments and enhanced carotenoids and anthocyanin [33].Delftia lacustris is potential as a plant growth-promoting bacteria (PGPB) [34]; [35], including the ability to release phytohormone, produce siderophore, and help plant resistance to pathogens [36]; [2].The phytohormones production by the Delftia lacustris supports the development of root, and plant production [37], and the gibberellin (Ga.3) hormone improves plant stem growth and fruit and also seed germination [7].The IAA is a useful hormone for cell diffraction and extension.Zeatin is a type of cytokinin hormone be useful for improving plant resistance to abiotic and biotic stress [38], and extending the dormancy period of shoots, tubers, and seeds [39] and Kinetin is another kind of cytokine hormone that plays a role in the propagation and development of shoots, increasing chlorophyll synthesis, and root growth [40]; [41].Notes: Nd = not detected

Liberica coffee growth on peat inoculated with cellulolytic bacteria
The average vegetative growth (plant height, stem circumference, and leave number) of the Liberica coffee seedlings growing on peat, inoculated, and uninoculated with cellulolytic bacteria was significantly different for all parameters (Table 7).On the other hand, the cellulolytic bacterial colonies inoculated on peat significantly influenced the growth of Liberica coffee seedlings.As mentioned before, the cellulolytic bacteria, Comamonas testosteroni and Delftia lacustris colonies enriched on peat could improve the availability of P by dissolving fixed P and also released some phytohormones such as auxins (IAA), gibberellin, zeatin, and kinetin.That all improved the Liberica coffee seedling growth (Fig. 2).Improving available P was essential for resistance to root, fruit, bean, and plant disease [42].Phosphate stimulates root development and strengthens stalk and stem [43].Whereas the release of IAA is essential for cell diffraction and extension, the gibberellin (Ga.3) for stem growth of plants, and seed germination [7].Zeatin, a cytokinin hormone, improves plant resistance to abiotic and biotic stress [38], and kinetin, another cytokine hormone supports the propagation and development of shoots, synthesis of chlorophyll, and root growth [40]; [41].
Enrichment of peat with indigenous cellulolytic bacteria, which can improve the growth of Liberika coffee through its ability to dissolve fixed P and release some phytohormones, is a signal that the enrichment of indigenous cellulolytic bacteria on peatland is an alternative technology towards a sustainable peatland management system.

Table 2 .
Cellulolytic activities index (AI) and cellulase activity of selected cellulolytic bacteria

Table 3 .
Bacterial species closely related to the cellulolytic bacteria of Jambi peat soils

Table 4 .
The ability of cellulolytic bacteria in the dissolution of phosphate

Table 5 .
The phytohormones produced by cellulolytic bacteria of peat