Isolation of bacteria with plant growth-promoting properties from microalgae-bacterial flocs produced in high-rate oxidation ponds

Figures & data

Figure 1. Scanning electron micrographs showing cell morphology of the twelve bacterial isolates obtained from HRAOP MaB-flocs. ECCN 1b (A); ECCN 2b (B); ECCN 3b (C); ECCN 4b (D); ECCN 5b (E); ECCN 6b (F); ECCN 7b (G); ECCN 8b (H); ECCN 9b (I); ECCN 10b (J); ECCN 11b (K) and ECCN 12b (L).

Table 1. Morphological and biochemical characteristics of bacterial colonies of isolates obtained from MaB-flocs formed in the HRAOP of an IAPS supplied municipal sewage.

Bacterial strain	Morphological characters		Biochemical characters
	Colony colour	Colony diam. (mm)	Gram-stain	Catalase
ECCN 1b	Orange-Brown	1–2	+	+
ECCN 2b	Creamy Yellow	1–4	+	+
ECCN 3b	Creamy White	1–2	+	+
ECCN 4b	White	1.5–2	−	+
ECCN 5b	Orange	1–2	+	+
ECCN 6b	White	1–2	+	+
ECCN 7b	Creamy White	2	−	+
ECCN 8b	Orange	2	+	+
ECCN 9b	Orange-Brown	3	+	+
ECCN 10b	Creamy White	1–2	−	+
ECCN 11b	Creamy White	≤1	−	+
ECCN 12b	Orange-Brown	1–2	+	+

Table 2. Molecular identity of bacteria isolated from MaB-flocs formed in HRAOP of the Belmont Valley IAPS supplied municipal sewage.

EBRU culture collection No.	Blast prediction of bacterial strains	Identity (%)	GenBank Accession No.	Length (bp)
ECCN 1b	Bacillus sp.	99	MW672568	1420
ECCN 2b	Fictibacillus sp.	100	MW672569	1471
ECCN 3b	Bacillus sp.	100	MW672570	1510
ECCN 4b	Aeromonas sp.	99	MW672571	1533
ECCN 5b	Exiguobacterium sp.	99	MW672573	1529
ECCN 6b	Arthrobacter sp.	100	MW672575	1522
ECCN 7b	Enterobacter sp.	99	MW672577	1389
ECCN 8b	Exiguobacterium sp.	99	MW672579	1504
ECCN 9b	Microbacterium sp.	98	MW672580	1488
ECCN 10b	Pseudomonas sp.	100	MW672582	1547
ECCN 11b	Ancylobacter sp.	100	MW672584	1428
ECCN 12b	Microbacterium sp.	91	MW672585	1486

Figure 2. Dendrogram showing the genetic relationship between the twelve potential PGPR strains (♦) isolated from MaB-flocs generated in HRAOP and reference strains contained in the GenBank database. Assigned strains and GenBank accession numbers are located to the right of each strain. Bootstrap test (1000 replicates) above branches, bootstrap values ≥56% at nodes and bar, substituting 5 nucleotides per 100.

Figure 3. Potential plant growth regulator production and essential nutrient mineralization by bacterial strains isolated from MaB-flocs produced in mixed liquor of the HRAOP of an IAPS supplied municipal sewage. Estimates of indole production (A); ammonium production (B); solubilised phosphate (C); and, solubilised potassium (D) by these bacterial isolates in stationary phase (OD₆₀₀ = 0.4 at 30°C) were determined spectrophotometrically. Results are the mean ± S.D. Means denoted by a different letter indicate significant differences (p < 0.05).

Table 3. Ranking of novel bacterial strains according to PGP activity potential. Strains were isolated from MaB-flocs in mixed liquor of the HRAOP of an IAPS supplied municipal sewage.

Rank	Species	Production/Solubilisation Activity
		IAA (equiv)	NH4Cl (equiv)	Soluble ${\mathrm{PO}}_4^{3-}$	Soluble K^+
1	Enterobacter strain ECCN 7b	+ +	+ +	+ +	+ +
2	Aeromonas strain ECCN 4b	+ +	+ +	+ +	– –
3	Arthrobacter strain ECCN 6b	+ +	+ –	– –	– –
4	Bacillus strain ECCN 3b	+ –	+ +	+ +	– –
4	Pseudomonas strain ECCN 10b	+ –	+ +	+ +	– –
5	Bacillus strain ECCN 1b	– –	+ +	+ +	+ –
5	Exiguobacterium strain ECCN 5b	– –	+ +	+ +	+ –
6	Exiguobacterium strain ECCN 8b	– –	+ +	+ +	– –
7	Fictibacillus strain ECCN 2b	– –	+ +	– –	– –
8	Microbacterium strain ECCN 12b	– –	– –	– –	+ –
9	Microbacterium strain ECCN 9b	– –	– –	– –	– –
9	Ancylobacter strain ECCN 11b	– –	– –	– –	– –

Note: Results are from a one-way ANOVA on ranks (where, + + is good; + – is intermediate; – – is low activity) using the Kruskal-Wallis H Test (P < 0.050).

Figure 4. Adventitious root formation in mung bean hypocotyls by cell-free filtrates from Aeromonas ECCN 4b, Arthrobacter ECCN 6b, and Enterobacter ECCN 7b grown in L-trp-supplemented medium. Indole production, measured as IAA equivalents, as a function of L-trp concentration (A); adventitious roots formed in mung bean hypocotyls treated either with IAA or cell-free bacterial filtrate (B); kinetics of adventitious root formation in mung bean hypocotyls treated either with IAA or cell-free bacterial filtrate (C); and, fresh and dry mass of the adventitious roots formed (D). Results are the mean ± S.D. Means followed by ** indicate significant differences from control + and the cell-free bacterial filtrate treatments (p < 0.05).

Figure 5. Fresh and dry mass of mung bean hypocotyls treated either with IAA or cell-free culture filtrates from L-trp-supplemented cultures of Aeromonas ECCN 4b, Arthrobacter ECCN 6b, and Enterobacter ECCN 7b. Initial fresh mass (FMt₀) was determined at start of the experiment (t₀) while final fresh mass (FMt₁₀) and dry mass (DMt₁₀) were determined after a 10-d incubation period (t₁₀). Results are the mean ± S.D. (p < 0.05).

Data availability

Data will be made available on request.