Exploration of IAA Producing Bacteria And Amylolitic Bacteria From Several East Java Lakes, and Their Potency For Microbial Consortium To Accelerate Chlorella Vulgaris Growth

The consortium of various types of bacteria from lakes in East Java has the potency to stimulate microalgae Chlorella vulgaris growth. Increased microalgae density from co-culture has an excellent potency for sources of biomass that can be developed for renewable energy. Several stages conducted of this research started from an exploration of IAA producing bacteria and amylolytic bacteria from several East Java Lakes; then, the highest bacterial isolates were identied with morphological and genotypical characteristics. The well-characterized bacterial isolates were used for the microbial consortium in co-culture with C. vulgaris. The treatment used in this study as follows: I) C. vulgaris without bacteria culture as a control, II) amylolytic bacteria + C. vulgaris, III) IAA-producing bacteria + C. vulgaris, IV) potential amylolytic bacteria and IAA-producing bacteria + C. vulgaris. The exploration result of potential bacteria from Ranu Pani, Ranu Regulo, Telaga Ngebel, and Ranu Grati lakes was found 53 amylolytic bacterial isolates, and 90 isolates IAA-producing bacteria. The highest amylolytic bacteria (isolate L) is related to Bacillus amyloliquefaciens, while the most elevated IAA-producing bacteria (isolate C) is related to Bacillus paramycoides. The highest cell density was produced in treatment III, reaching 2.7 x 10 6 cells/mL on day 50th. The treatments with supplement bacteria showed a signicant effect for accelerating the growth of microalgae compared to control.


Introduction
Energy dependence and continuous energy consumption could cause an energy crisis. Global demand for petroleum is expected to increase by 40% in 2025, so environmentally friendly alternative fuels are needed (Hirsch et al., 2005). Biofuel from biological sources (biomass) has a potential resource for renewable energy playing an essential role in the future global energy infrastructure (Cheah et al., 2020;Tandon et al., 2017). Biomass from microalgae has been considered one of the most promising raw materials as a renewable energy source because not producing emissions in their process to generate biofuels (Cheah et al., 2020;Chisti, 2007;Tandon et al., 2017). The recent studies with culture C. vulgaris showed the advantages of biomass production compared with plants (Tandon et al., 2017). Besides, C. vulgaris has rapid growth correlated with numerous lipid production, low cost, and does not consume large scale tracts of land (Chisti, 2007;Tandon et al., 2017).
The problem in the monoculture of microalgae is how the high biomass with low cost and a short period can be obtained. Single-species cultivation for microalgae is challenging for biomass production on a large scale because it is contrary to the natural tendency of an ecosystem to become increasingly complex (Kazamia et al., 2012). Hence, the use of a microbial consortium consisting of several kinds of bacteria and microalgae can optimize the growth of microalgae in co-culture (Kazamia et al., 2012). This microbial consortium with various types of bacteria from lakes in East Java has a great potential to optimize culture condition; this condition is essential for large-scale cultivation. Co-culture between microalgae and bacteria can increase the microalgae growth because those bacteria are providing essential inorganic mineral elements for microalgae (Marañón et al., 2005). The blooms of algae and aquatic plants are inseparable from the in uence of decomposing bacteria in the water environment because decomposing bacteria plays as decomposers of organic components into simpler components as nutrients.
Ranu Pani and Ranu Regulo have an average organic substrate content of water, about 18.18% and 26.06%, respectively (Gazali et al., 2014). Ranu Grati has a pH of 7.67 with average temperatures of 29.07°C and a fertility value of 52.41. Based on the TSI value, Ranu Grati classi ed it as freshwater that had experienced mild eutrophication. Organic materials in the four lakes come from microalgae, plant parts, micro-tricks, agriculture, and waste. The high content of dissolved organic matter (DOM) is an indicator of the abundance and richness of microbes (Gazali et al., 2014).
Amylolytic bacteria can stimulate the microalgae growth by producing amylase enzymes to break down amylum into simple sugars, like glucose, through several stages (Gazali et al., 2014;Silaban et al., 2020).
Those simpler molecules can be directly used for supporting microalgae growth. Amylase enzymes from bacteria are widely used in various industries because the strain has rapid growth and require a short amount of time to produce amylases (Keeney & College, 2007). In general, those enzymes were used in textile industries, food industries, and pulp industries. However, those enzymes can also be used in microalgae culture because this type of bacteria can degrade substrates e ciently then be utilized by algae for its growth (Keeney & College, 2007;Souza & Magalhães, 2010).
On the other hand, bacteria producing indole-3-Acetic Acid (IAA hormone) can provide growth factors for responses to light stimulation in plants, including microalgae (Zhao, 2010). IAA is the exogenous auxin class produced by bacteria through the dependent pathway with the addition of tryptophan and independent pathway without tryptophan (Nonhebel, 2015). Most bacteria produce IAA with dependent pathways, notably in the indole-3-acetamide (IAM) pathway and the indole-3-pyruvate (IPA) pathway (Ozdal et al., 2017). IAA hormone is essential for the growth of microalgae, especially C. vulgaris (L. De-Bashan, 2008) due to that hormone regulates the growth process and induces the production of metabolites. The utilization of a controlled consortium of bacteria and microalgae could increase microalgae biomass production (Chisti, 2007

Samples collection
Water samples were collected from several lakes in East Java, Indonesia (listed in Table 1) with ve 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. 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-modi ed 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 modi ed-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: Amylum hydrolysis index data obtained were then analyzed using ANOVA.

Modi cated-Gusrina Media preparation
The Gusrina media that has been modi ed was made by 45 ppm urea, 30 ppm TSP (Triple Super Phosphate), amount of 1 ppm FeCl 3 , and 25 µg / L of vitamin B12 (Hadiyanto et al., 2012). Those components dissolved with distilled water up to 1000 mL into an Erlenmeyer ask 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 20 0 C for 24 h. These bacteria were measured the cell density using the 0.5 Mac. Farland solution scale that has equivalent to 1.5 × 10 8 cfu/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 modi ed 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.

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 20 0 C. The collected data were presented in a graphic, and they were analyzed with one-way ANOVA. A 5% LSD followed the signi cance test to determine the best treatment.

Environmental conditions from Lakes
The measured abiotic factors from each lake and station were already listed in Table 3. The total isolates of IAA-producing bacteria and produced IAA content Ninety isolates of bacteria that positively produce IAA from Ranu Grati, Ranu Pani, Ranu Regulo, and Telaga Ngebel were successfully isolated. As a result, the ten highest bacterial isolates based on UV-Visible spectrophotometer measurements were recorded in Table 4, and complete data were listed in (supplementary les 1). The bacterial isolate C from Ranu Pani was able to produce the highest IAA hormone concentration of 158.11 ppm. The total amylolytic bacteria and their activity Fifty-three bacterial isolates from Ranu Pani, Ranu Regulo, Ranu Grati, and Telaga Ngebel had been known to be amylolytic. The average results of calculating the most extensive ten amylum hydrolysis index can be seen in Table 5, and full data can be seen in (supplementary les 2). Table 5 shows that the L bacterial isolate from Ranu Pani had the highest hydrolysis index value of 5.89. Bacterial isolate C and L identi cation The morphological characteristics of isolate C and isolate L can be seen in Table 6. Based on those morphological characteristics, bacterial isolate C and bacterial isolate L could be described as genus of Bacilli; but, the annotation to level species cannot be determined.
To inform identi cation results based on barcoding DNA with 16S rRNA sequence, in this paper, we were re-analysis 16S sequence from (Rodiansyah et al., 2021) with additional reference sequences speci c on Bacillus cereus Group and Bacillus substilis Group. The maximum likelihood (ML) tree showed that both genus Bacilli separated into two groups with a con dence value of 100. The bacterial isolate C is located in one clade with Bacillus paramycoides with a con dence value of 66. However, the bacterial isolate L was situated in the same clade with Bacillus amyloliquefaciens with a bootstrap value of 36 (Fig. 1).
The genetic distance analysis from genus Bacillus shows a signi cant difference in sequences betweengroup similarity, about 94%, but within-group similarity shows they are homogeny (Similarity 99%) ( Table 7).

Chlorella vulgaris cells density and data analysis
The graph in Fig. 2 below shows the in uence of the treatments on the microalgae growth from day 0 to 50. The growth data was measured with the calculation of cell density using haemocytometer. In general, microbial consortium treatments have signi cantly different growth of microalgae compared to control.
In treatment I (control treatment), the pattern of the growth of C. vulgaris start from the lag phase (day 0 to 6), then the log phase (day 6 to 12), and nally, the death phase (day 12 to 50) (purple line in Fig. 2). The graph of the microalgae growth in this treatment has a moderate increase from day 2 to 12 that has the highest average cell density reaching 3.5 x 10 5 cells/mL. After that, the growth of microalgae decreased gradually on day 14 and then levelled off until day 50 that has an average cell density of 3 x 10 3 cells/mL.
In treatment II, the growth of C. vulgaris starts from the lag phase (day 0 to 10), log phase (day 10 to 38), and death phase (day 40 to 50) (green line in Fig. 2). The growth of microalgae in the lag phase decreased slightly from day 0 to 10, in the log phase rose gradually and then climbed sharply, with an average cell density of 2.4 x 10 6 cells/mL. After reaching the death phase, the growth of microalgae dropped moderately, with an average cell density of 1.9 x 10 6 cells/mL. In treatment III, the development of C. vulgaris start from the lag phase (day 0 to 8); after that, the growth of microalgae fell steadily until day ten, then boom dramatically at the log phase (day 12 to 50) with cells density reaching at 2.7 x 10 6 cells/mL (red line in Fig. 2). In treatment IV, the growth of C. vulgaris starts from the lag phase (day 0 to 10), log phase (day 12 to 38), and death phase (day 40 to 50) (blue line in Fig. 2). At the lag phase, the growth of microalgae decreased slowly then climb dramatically at the log phase with a cell density of 2.1 x 10 6 cells/mL; nally, there was a fall gradual in the death phase with an average cell density of 1.4 x 10 6 cells/mL.
The hypothesis calculated with ANOVA t-test shows that te F value > F Table (Table 8); as a result, the hypothesis is accepted, indicated that there is an in uence of a consortium of amylolytic bacteria and IAA producing bacteria on the C. vulgaris growth. The treatments other than control gives a signi cant in uence on the development of the density of C. vulgaris cells. The further test with the Least Signi cant Difference Test (LSD) signi cant 5% can be seen in Table 8. Treatment III showed the best treatment for increasing C. vulgaris, which microbial consortium treatments are signi cantly different compared to the control treatment (Table 9).

Discussion
Heterotrophic bacteria are available in various ecosystems, including lake waters (K. Liu et al., 2016). The presence of heterotrophic bacteria in lake waters is in uenced by organic material in lake waters that can support bacterial growth (Yuningsih et al., 2014). Heterotrophic bacteria can decompose complex organic compounds containing elements C, H, and N into simpler compounds because they can produce extracellular enzymes (K. Liu et al., 2016). Some bacterial isolates that were successfully isolated in this study prove that the waters from four lakes can supply the nutrition needed by bacteria for supporting their life.
Amylolytic bacteria can gradually degrade amylum in lake waters because amylum can not directly convert amylum into glucose (Rahmansyah & Sudiana, 2010). Initially, amylum will be hydrolyzed into the dextrin compound in the form of polysaccharides. The dextrin is re-hydrolyzed again into oligosaccharides in the form of maltose. Maltose is subsequently converted into monosaccharides in the form of glucose (Brewster, 1953). Our highest amylolytic bacteria from Ranu Pani, based on phenotypic and genotypic characteristics, was identi ed as Bacillus amyloliquefaciens. This species is an amylolytic bacteria that play an essential role as a remodel of organic materials into simpler inorganic components in nature (Waluyo, 2009). Amylolytic bacteria can produce extracellular enzymes to degrading amylum in water (Brown et al., 2008). Amylum that has been degraded into simpler compounds can be utilized by C. vulgaris microalgae as a raw material for the photosynthesis process (Brown et al., 2008). The carbon element from carbohydrate and protein decomposition by bacteria in waters can increase the biomass of microalgae C. vulgaris (Brown et al., 2008). The essence of carbon can be assimilated in waters in the form of CO 2 , which plays a role in the photosynthetic process of microalgae C. vulgaris (Hopkin, 2020).
On the other hand, this study also uses the highest potential bacteria producing IAA from Ranu Pani, identi ed as Bacillus paramycoides. The analysis results of variants obtained that IAA-producing bacteria signi cantly in uence the growth of C. vulgaris. Further tests explained that the treatment of a consortium of IAA-producing bacteria had the most signi cant in uence on the cell density of C. vulgaris as the indicator from rate growth. Bacteria produce IAA hormones through gene regulation and tryptophan biosynthesis in microalgae as a precursor for bacteria regulation. Amino acids, such as tryptophan, are easily detected by pairs of organisms in the culture to act as nutritional signals for synthesis IAA with indole 3-glycerol phosphate as the primary substrate (Hopkin, 2020;Vessey, 2016). They live together in waters as mutualism symbiotic. Auxin produced from these bacteria could encourage microalgae cell division processes to increase microalgae biomass (Cooper & Smith, 2015). Bacillus paramycoides play a vital role in the decomposition of organic compounds that have a function in the growth of microalgae (Vessey, 2016). This species can hydrolyze amylum, produce H 2 S, urease, tryptophan deaminase, indole production, and acid production from mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, and arabinose (Y. Liu et al., 2017). These multiple potencies from Bacillus paramycoides showed probably more e cient for stimulating the C. vulgaris growth because that bacterium continues to produce IAA in a long period at culture media (Pratt, 1938).
In the microbial consortium, bacteria can reduce the oxygen pressure in the culture, and they can convert complex elements (N, S, P, and C) to become elements ready consumed for algae photosynthesis

Conclusions
The most potential IAA-producing bacteria identi ed as Bacillus paramycoides from Ranu Pani with an IAA concentration of 158.11 ppm, while the highest amylolytic bacteria has a hydrolyze index of 5.9 identi ed as Bacillus amyloliquefaciens. The microbial consortium shows the effectiveness of stimulating C. vulgaris growth compared to control. The microbial consortium with IAA-producing bacteria supplementation was the most effective to enhance microalgae growth, reaching up to 2.7 x 10 6 cells/mL of cell density on day 50. Future research with broad-scale culture in bioreactor and exploring the speci c interaction in co-culture are needed for obtaining high biomass. Figure 1 Phylogenetic tree of C isolate, and L isolate with ML method. Salmonella enterica subs. enterica Strain LT2 and Ty2 set as out of the group.