Samples collection
Water samples were collected from several lakes in East Java, Indonesia (listed in Table 1) with five stations from each lake. Approximately 1000 mL water samples were stored in a sterile bottle; then, placed into the ice-box to protect the pieces from degradation until the laboratory works.
Table 1
List of sampling location.
No
|
Lakes
|
Location
|
Altitude
|
1
|
Ranu Pani
|
Lumajang regency
|
2.114 masl
|
2
|
Ranu Regulo
|
Lumajang regency
|
2.100 masl
|
3
|
Ranu Grati
|
Pasuruan regency
|
100 masl
|
4
|
Telaga Ngebel
|
Ponorogo
|
745 masl
|
Abiotic factors were also measured in this study. Those factors include light intensity using Lux meter SANFIX (LX-1330 B), water temperature, and pH were measured using Refractometer ATAGO S-28E, dissolved oxygen (DO) using oxygen meter (Lutron DO-5510), pH meter Lutron 5509, respectively. Water turbidity was measured using a turbidity meter (TOA-TB 25A).
Isolation of IAA producing bacteria and quantification IAA contents
Single colonies that grew with different characteristics were moved into a new plate that contains TSA media with additional tryptophan (25:1). The positive bacterial isolates to produce IAA were tested qualitatively by the colourimetric method using the Salkowski reagent. This reagent was made by 1 mL of 0.5 M FeCl3 (1.35 g / 10 mL) and 50 mL of 50% HClO4 (25 mL HClO4 + 25 mL H2O) then the reagent was stored in an untranslucent bottle at room temperature. Single isolates presenting a pink colour after dropped Salkowski reagent indicated they have the activity to produce IAA. Those positive isolates were used for quantitative tests using UV-vis spectrophotometry.
Single isolates from TSA media were cultured on Tryptic Soy Broth (TSB) media with an additional tryptophan (200 ppm) with a ratio (25:1). The spectrophotometric method was used for quantifying IAA contents produced by the isolates. The concentration of IAA was analyzed based on the absorbance value absorbed by UV-Visible spectrophotometer compared to the IAA standard curve. The absorbance value was directly proportional to the IAA standard curve and IAA produced by the isolates. The standard curve was made using pure IAA. The wavelength used was 530 nm; this wavelength was chosen based on the colour produced by the interaction between the Salkowski reagent and the IAA (Glickmann & Dessaux, 1995).
Amylolitic bacteria selection and semiquantitative assay of amylolytic bacteria activity
The growth medium was used for the single colonies selection is peptone media 0.1% and Plate Count Agar (PCA) media (instant PCA powder 23.5g / L from Merck, USA). The bacteria from water samples were cultured using the serial dilution method with 0.1% peptone buffer then spread into PCA media. Colonies that grew in PCA media were selected based on different colony morphologies to get the single isolates. NA-modified media that was composed of instant nutrient agar (NA) powder 20g / L (Merck, USA) with an additional 2g of amylum (Sigma-Aldrich, USA) was used for selecting and calculating the hydrolysis index from amylolytic bacteria. These bacterial isolates will produce a clear zone around the colony after dropped the iodine. The bacterium sample was aseptically inoculated into modified-NA media using the quadrant method. The amylum hydrolysis index of each isolate can be determined based on the diameter of the clear zone formed on the amylum agar medium.
The formula for calculation the amylum hydrolysis index is as follows:
![](data:image/png;base64,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)
d = Diameter
Amylum hydrolysis index data obtained were then analyzed using ANOVA.
Modificated-Gusrina Media preparation
The Gusrina media that has been modified was made by 45 ppm urea, 30 ppm TSP (Triple Super Phosphate), amount of 1 ppm FeCl3, and 25 µg / L of vitamin B12 (Hadiyanto et al., 2012). Those components dissolved with distilled water up to 1000 mL into an Erlenmeyer flask then heated with a hot plate. This media stock was made with 10× concentration then it can be stored in the refrigerator.
Bacterial stock culture and algae stock culture for microbial consortium
The culture of the highest amylolytic bacteria and the highest ability of IAA-producing bacteria were cultured in 15 mL of nutrient broth (NB) medium at 200C for 24 h. These bacteria were measured the cell density using the 0.5 Mac. Farland solution scale that has equivalent to 1.5 × 108cfu/mL. These bacterial cultures conditions were used as a starter in co-culture.
The microalgae strain that was used in this study is C. vulgaris (Purchased from LIPI, Indonesia). The stock of C. vulgaris was made with a ratio of 1:4 between the cultures and the medium. The 2,000 mL of the C. vulgaris stocks, which have optimal growth, were moved into a plastic tank with capacity up to 15,000 mL, then 10 mL of the Gusrina medium and distilled water up to 10,000 mL were added. After in exponential phase, 1,000 mL of stocks were divided into 20 tank glasses. These tank glasses were used for the consortium in co-culture C. vulgaris.
Consortium condition in microbial consortium with co-culture C. vulgaris
The medium used for co-culture C. vulgaris contains 0.5 mL of Gusrina modified media, 50 mL stock of microalgae, and 400 mL of sterile water. Those solutions for the co-culture medium were placed into a closed-tank glass that has a capacity of 1,000 mL. The treatments were used in co-culture can be seen in Table 2.
Table 2
The treatments used in this study.
Treatments
|
Composition
|
I (control)
|
50 mL C. vulgaris without the bacteria
|
II
|
50 mL C. vulgaris + 50 mL amylolytic bacteria
|
III
|
50 mL C. Vulgaris + 50 mL IAA-producing bacteria
|
IV
|
50 mL C. vulgaris + 25 mL amylolytic bacteria + 25 mL IAA-producing bacteria
|
The co-culture was given an aerator set (12 h aerator: 12 h without aerator) and lighting time (16 h lighting: 8 h without light exposure) (Hadiyanto et al., 2012; Lirofiatillah et al., 2020). Those treatments were repeated five times then placed in a closed room at a light intensity of 100–200 µE sec− 1 m− 2, 25°C.
Chlorella vulgaris cells density calculation and data analysis
Calculation of C. vulgaris cells density was using a hemocytometer. The observation was conducted every two days until the 50th days. The bacterial growth was observed every three days. The 50 µL sample was taken from the tank; then, using a dilution technique, the samples were diluted to reach a dilution level of 10− 6. At each dilution of 10− 4, 10− 5, and 10− 6, was taken 100 µL, then put into a petri dish containing PCA media, incubated for 24 h at 200 C. The collected data were presented in a graphic, and they were analyzed with one-way ANOVA. A 5% LSD followed the significance test to determine the best treatment.