Isolation and Identification of Photosynthetic Bacteria

Supplementary Material

Isolation and Identification of Photosynthetic Bacteria

Sediment samples from a pond of grass carp in the Zhejiang province of China were collected. The excess sediment sample is added in the sodium acetate-basal medium (Table 1),which was according to Van Niel (1971) and Qian & Huang (1999). The mixture was anaerobic culture in intelligent light incubator (ZGX-300C,Hangzhou Qianjiang equipment Co., Ltd.) at 5 ℃and light intensity 3000 lx. Anaerobic medium was prepared usingHungate anaerobic culture tubes (Hungate, 1968).When culture medium was reddish brown, the reddish brown culture medium was inoculated three or four times into a fresh medium. After which the bacteria were diluted and separated on the isolated medium plate closed with the Sealing film. The isolation medium (Table 2) was according to RCVBNmedium (Weaver, et al., 1975) and Rhodospirillaceae isolation medium (Qian and Huang, 1999).The plate was placed in the light till red colonies appeared. A single strain was continuously isolated three times from the red colonies. The isolate was identified as purple non-sulfur bacteria Rhodopseudomonas palustris through morphological feature, biochemical characteristics and 16S rDNA sequence analysis.

Table 1 The composition of a sodium acetate-basal medium

Component / Amount (l-1) / Component / Amount(l-1)
Sodium acetate (g)
NH4Cl (g)
MgSO4·7H2O (g)
K2HPO4 (g)
NaCl (g)
CaCl2·2H2O (g)
NaHCO3 * (g)
Mineral solution+ (ml)
Vitamin solution++ (ml)
Distilled water (ml)
pH Value / 1.0-5.0
1.0
0.2
0.2
0.5-2.0
0.05
1.0
10.0
10.0
980.0
6.8-7.2 / +Mineral solution
FeCl3·6H2O (g)
ZnSO4·7H2O (g)
H3BO4 (g)
CuSO4·5H2O (mg)
MnCl2·4H2O (mg)
Co(NO3)2·6H2O (mg)
++Vitamin solution
p-Aminobenzoic acid (mg)
Thiamine-HCl (mg)
Nicotinic acid (mg)
Biotin (mg) / 0.05
0.01
0.01
0.50
0.50
5.00
0.1
0.001
0.1
0.001

Note: * Filtration sterilization

Table 2 The composition of differential medium

Component / Amount (l-1) / Component / Amount(l-1)
NH4Cl (g)
MgSO4·7H2O (g)
K2HPO4 (g)
NaCl (g)
NaHCO3 a (ml)
Ethanol b (ml) / 1.0
0.2
0.5
2.0
20.0
2.0 / Yeast extract (g)
Agar (g)
Na2S·9H2O (g)
Distilled water (ml)
pH Value c / 2.0
2.0
1.0
978.0
7.0

Note: a Filtration sterilization (NaHCO3 0.1g/ml); b Filtration sterilization; c Adjust to pH 7.0 using phosphoric acid. (0.1 mol/L H3PO4, filtration sterilization)

Fig.S1 Effect ofprobiotics as water additives on the water quality in grass carp culture.

Fig.S2Cluster analysis of the water microbiome sequencing data of controls and treatments at genus level by Ward method.

Table S1Primer and sample-specific barcode sequences

Primer / Barcode sequence / Primer sequence
V3-20F / ATGAGAGT / ATGAGAGTCCTACGGGAGGCAGCAG
V3-20R / ATGAGAGTATTACCGCGGCTGCT
V3-21F / ATGAGTAG / ATGAGTAGCCTACGGGAGGCAGCAG
V3-21R / ATGAGTAGATTACCGCGGCTGCT
V3-22F / ATGAGTGT / ATGAGTGTCCTACGGGAGGCAGCAG
V3-22R / ATGAGTGTATTACCGCGGCTGCT
V3-29F / ATGTCGAT / ATGTCGATCCTACGGGAGGCAGCAG
V3-29R / ATGTCGATATTACCGCGGCTGCT
V3-30F / ATGTCGTG / ATGTCGTGCCTACGGGAGGCAGCAG
V3-30R / ATGTCGTGATTACCGCGGCTGCT
V3-31F / ATGCTATG / ATGCTATGCCTACGGGAGGCAGCAG
V3-31R / ATGCTATGATTACCGCGGCTGCT

Supplementary references

Qian, C.R., Huang, Y.X. (1999) Laboratory experiments in Microbiology. Peking University Press, Beijing. pp211-212.

Weaver,P.F.,Wall,J.D., Gest, H. (1975) Characterization of Rhodopseudomonas capsulata. Arch Microbiol105, 207-216.

Hungate, R.E.(1968) A roll tube method for cultivation of strict anaerobes. In: Morris & Robbins D.W. (Eds.), Methods in Microbiology, Vol. 3. Academic Press, London. pp117-132.

Van Niel, C.B.(1971) Techniques for the enrichment, isolation and maintenance of the photosynthetic bacteria. In: San Pietro A. (Eds.), Methods in Enzymology. Axademic Press, London. pp3-28.