Nafisah W, Prabaningtyas S, Witjoro A, Saptawati RT, Rodiansyah A. 2022. Exploration non-symbiotic nitrogen-fixing bacteria from several lakes in East Java, Indonesia. Biodiversitas 23: 1752-1758. Potential microorganisms, mainly nitrogen-fixing bacteria spread in almost lake areas in East Java. The biogeochemical processes that occur between biotic-biotic or biotic-abiotic factors can accelerate the growth of autotrophic microorganisms in waters as like microalgae. Non-symbiotic nitrogen-fixing bacteria in the water can supply nitrogen to support the growth of microalgae. This study is aimed to explore and calculate the ammonium concentration produced by non-symbiotic nitrogen-fixing bacteria that isolated from several lakes in East Java. The highest ammonium producing bacteria were characterized based on morphology, physiology, and genetic with 16S rRNA gene sequencing. The bacterial isolates were screened with a nitrogen-free bromothymol blue medium to select bacterial isolates capable of fixing nitrogen. The content of NH3/NH4 was produced by isolates of nitrogen-fixing bacteria was measured by using the prodac test kit NH3/NH4. The ammonium concentration was calculated with Nessler’s reagent by using the spectrophotometry method at a wavelength of 425 nm. The result was obtained 31 isolates of non-symbiotic nitrogen-fixing bacteria. The bacterial isolate B2 exhibited highest activity of fixing nitrogen that produced ammonium (14.46 mg/L) among the other screened bacteria. The morphological and physiological characterization from that isolate was identified as genus Bacillus, whereas the genetic characterization similar to Bacillus paramycoides, that included in the Bacillus cereus group.

Nisa IK, Prabaningtyas S, Lukiati B, Saptawati RT, Rodiansyah A. 2021. The potential of amylase enzyme activity against bacteria isolated from several lakes in East Java, Indonesia. Biodiversitas 21: 42-49. Indonesia is one country that has water resources having an abundance of microbial diversity, but not explored massively. This study aims to measure the amylase activity quantitatively from 53 amylolytic bacterial isolates from Ranu Pani, Ranu Regulo, Ranu Grati, and Ngebel Lake; also it identifies the isolate with the highest amylase enzyme activity. The amylase enzyme activity test calculates with DNS (Dinitrosalycylic acid) method, molecular identification of the highest bacterial isolate is based on the 16S rRNA gene. Its relationship is determined through the phylogenetic tree with the Neighbor-Joining (NJ) method. The results showed that the fifty-three bacterial isolates have amylase activity about 0.000-0.016 units/mL. The KN bacterial isolate from Ranu Ngebel was the highest amylase activity, producing enzyme around 0.016 units/mL, while isolate G20 from Ranu Grati was the lowest, reaching about 0.0001 Unit/mL. Based on the morphological and molecular identification, the KN bacterial isolate is classified as the Bacillus cereus group with 99.4-100% sequence similarity, closely related to Bacillus paramycoides (NR 157734.1).

Prabaningtyas S, Ardyati T, Suharjono, Retnaningdyah C. 2021. Exploration of vitamin B12-producing bacteria from Indonesia Eutrophic Lake: A new strategy to improve microalgae biomass production. Biodiversitas 22: 4538-4544. Biofuel producing autotrophs and heterotrophs organisms are mostly found in freshwaters such as lakes. Preliminary research showed that Ranu Grati has a high diversity of microalgae and bacteria. Microalgae are known as one source of biofuels due to the high content of lipid. The growth of microalgae is strongly influenced by vitamin B12, which is synthesized by bacteria. The role of vitamin B12 in the metabolism of algae is mainly as a co-enzyme for vitamin B12-dependent methionine synthase. The purpose of this research was to observe the bacterial diversity based on NGS analysis, to explore vitamin B12 produced by bacteria from Ranu Grati lake, and also to identify the highest potency of vitamin B12-producing bacteria based on 16S rRNA gene sequencing. The study was carried out by taking a water sample at a depth of 50 cm at five stations. Isolation of total bacterial DNA was carried out using FastDNA Spin Kit for Soil. The metagenomic method of Next Generation Sequencing (NGS) was used as an initial study of bacterial diversity. Vitamin B12-producing bacteria was isolated using PYBG agar (phytone peptone, trypticase peptone, lab-lemco powder, bacto yeast extract, glucose, agar). Isolated bacteria were screened for production of vitamin B12. The potential isolates were identified base on 16S rRNA gene sequence similarity. The results of the metagenomic study showed that the genus of potential bacteria producing vitamin B12 included Bacillus, Pseudomonas, Propionibacterium, Enterobacter, Escherichia, Acinetobacter, and Flavobacterium. The results of screening with PYBG media obtained 30 isolates of vitamin B12 producing bacteria. Ten efficient vitamin B12-producing isolates were identified as Lysinibacillus fusiformis (isolate G2V1), Bacillus cereus group (isolate G2V25, G2V24, G2V13, G2V9, G2V8), Alcaligenes faecalis (isolate G2V22), Delftia acidovorans (isolate G2V14A) and the genus Delftia (isolate G2V19). The isolate G2V1 (Lysinibacillus fusiformis) was the highest producer of vitamin B12 which was able to produce 33,783 ug/mL of vitamin B12 at 4 days incubation time.

The need for energy sources in Indonesia continues to increase, in 2018 Indonesia is a country with energy consumption of around 114 Million Tons of Oil Equivalent (MTOE). Therefore, alternative energy from biomass such as biodiesel is needed. Chlorella vulgaris is a microbe suitable for alternative energy because it contains up to 80% lipids. Chlorella vulgaris needs phosphorus for cell growth from fertilizer for the production of Adenosine Triphosphate (ATP). In addition to phosphorus, co-cultures with IAA-producing bacteria can promote the growth of C. vulgaris. This study was conducted to evaluate the influence of phosphate sources on the growth of C. vulgaris cultured with IAA-producing bacteria. This study was carried out by co-culture C. vulgaris and IAA-producing bacteria with different phosphate sources on Gusrina medium for 27 days. The treatment consisted of treatment A (control), B Triple Super Phosphate (TSP), and C Super Phosphate-36 (SP-36). Cell growth was calculated using the Petroff-Hausser counting chamber method followed by the ANOVA test. The highest cell growth was obtained in treatment B (TSP), followed by treatment C (SP-36), and finally treatment A (control). The results showed that the addition of phosphate sources had an effect on the growth of C. vulgaris with the highest number of cells found in treatment B (TSP) of 2,66 × 106 cells/mL. Keywords: Chlorella vulgaris; co-culture; phosphorus

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 identified 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 10⁶ cells/mL on day 50th. The treatments with supplement bacteria showed a significant effect for accelerating the growth of microalgae compared to control.

Microalgae have the potential to be developed. As a source of biofuels, medicines, cosmetics, sources of food and feed, it is necessary to optimize microalgae growth to increase microalgae growth. Bacteria can increase microalgae growth through various mechanisms such as producing CO₂, degrading organic molecules, providing vitamin B12, and preventing oxidative stress. One important factor for the growth of microalgae is culture media. Therefore, it is necessary to do research to find the medium culture that gives the highest density of microalgae cells. The research objective was to determine the effect of the addition of bacteria and culture media on the growth of Chlorella vulgaris. Our research used microalgae-bacterial coculture in Guillard and Gusrina medium. The culture conditions is set 12 hours without an aerator, 12 hours with an aerator; 16 hours of light; 8 dark hours; light intensity 2700 lux. The results of the study showed the coculture treatment had higher cell density of about 3.8 × 10⁷ cells/mL and Gusrina medium showed the highest cell density at about 4.6 × 10⁷ cells/mL. The addition of bacteria was able to increase the growth of microalgae and Gusrina medium showed a good growth medium for C. vulgaris.