Developed biofilm-based biofertilizer as a bioremediation agent for agroecosystem and environment contaminated with Cr (VI)

Biofilm are microbial community that attaches to one substrate and another through EPS. Functional microbes in biofilm can be used as a biofertilizer which increase plant growth by providing nutrients and plant resistance to pathogens due to agricultural environmental degradation. Beside being a biofertilizer, biofilm can be developed as a bioremediation agent. Hexavalent chromium (Cr (VI)) is a heavy metal that is widely used in the leather tanning, pharmaceutical and metallurgical industries, so it is easily found in irrigation and causes agricultural land pollution. Chrome can be toxic to microorganisms, plants, animals and humans, because it is carcinogenic, causes ecosystem damage and has a negative impact on human health. Various techniques are used to remediate Cr (VI), one method that can be used is bioremediation by exploiting the potential of bacteri or fungi incorporated in the biofilm. In this study, the biofilm consisted of bacterial and fungi (BFBF) that were found on the western slopes of Mount Lawu. The Cr (VI) reduction test was carried out at concentrations of 5 and 50 mg L−1. The results showed that the biofilm was able to reduce Cr (VI) up to 1.19 mg L−1within 6 hours.


Introduction
Biofilms are microbial communities that adhere to one substrate and another through the production of extracellular polymeric substances [1].Biofilms in agricultural environments are used as biofertilizers which can reduce the use of inorganic fertilizer because biofilm consists of functional microbes that are able to increase nitrogen fixation, release of nutrients, organic acids and production of plant growth hormones [2].In this research, biofilm-based biofertilizer were formed from functional microbes such as nitrogen-fixing bacteria, phosphate solubilizing microbia and potassium solubilizing fungi.Beside being used as biofertilizer, biofilms in environments with high metal concentrations can play an important role for the plants above because they are able to immobilize contaminants and precipitate heavy metals.Biofilms are able to reduce metals such as Pb 2+ , Cd 2+ , and Zn 2+ and reduce metal penetration into plants such as potato tubers [3].The use of biofilm-based biofertilizer is one of the keys to healthy agriculture due to the high degradation of agricultural land.
Chromium is a heavy metal that often causes agricultural land degradation.Chromium is mostly applied in modern industries such as tanning, iron casting, pharmaceutical industries, metallurgical industry, textile dyes, paint industry, metal refining and others [4], [5] which are then disposed of inappropriately into irrigation flows so that they can enter agricultural land,

Materials and methods
This research was held in August 2023 at the Soil Biology and Biotechnology Laboratory, Faculty of Agriculture, UNS.The biofilm consisted of 4 bacterial isolates and 5 fungal isolates which were combined and then tested to determine the Cr (VI) reduction activity using bacterial culture treatment (growing cells).Biofilm growth curve testing was carried out by growing the biofilm on a combined PDB+NB medium and measuring it every 4 hours until the 88 th hour of observation, then the measured optic density of the biofilm growth using a spectrophotometer OD = 600 nm.
Cr (VI) reduction test wih biofilms were conducted on minimal media with low glucose levels.Each isolate was taken with a ose needle and inoculated into 10 ml of NB media for bacterial isolates and PDB for fungal isolates.The culture was incubated for 10 hours at room temperature and shaker at 90 rpm.0.5 ml of each culture was then taken (OD 600 nm = 1) and combined by inoculating to 20 ml of minimal medium (10 g glucose; KH2PO4 6.8 g; K2HPO2 8.7 g; (NH4)2SO2 3.3 g; MgSO4 1.23 g per liter of distilled water), then incubated for 20 hours at room temperature with shaking at a speed of 90 rpm.2.5 ml of culture was taken (OD 600 nm = 1) and inoculated in 250 ml of sterile minimal medium, incubated for 32 hours at room temperature with shaking at 90 rpm to reach the peak log phase.At the 52 nd hour it was stopped and Cr (VI) was added in the form K2Cr2O7 with concentrations of 5 and 50 mg L -1 and minimal media containing Cr (VI) according to the treatment without biofilm was used as a control and continued with incubation for 48 hours with the same conditions.Sampling was carried out every 6 hours to determine biofilm growth and residual Cr (VI) levels.Bacterial growth was measured for optical density at 600 nm using a spectrophotometer.Residual Cr (VI) levels in medium were determined using the diphenyl carbazide (DPC) method [11], [12].
One ml of liquid medium containing biofilm and Cr (VI) was taken and put into1.5 ml eppendof tube, and was centrifuged at 10.000 rpm in 5 minutes.A total of 0.4 ml samples were taken and the volume was made to 1 ml by adding distilled water.Then 0.33 ml 6M H2SO4 was added to the solution and 0.4 ml DPC (0.25% w/v).DPC 0.25% (w/v) was prepared by dissolving 0.125 g of diphenyl carbazide in 50 ml of acetone.Volume of the solution was increased to 10 ml adding distilled water, and the solution was allowed to react for 10 minutes.The sample was then measured with a double beam UV-VIS spectrophotometer at a wavelength of 540 nm [11], [12].

Result and Discuccion
Biofilms in agricultural ecosystems can be used as a substitute for the use of inorganic fertilizers.This is because biofilm-biofertilizer can improve the chemical, physical and microbiological properties of soil and stimulate plant growth [13].Beside being used as a biofertilizer, it is known that biofilm are known as a multifunctional biofertilizer which can act as a bioremediation agent, able to reduce the amount of heavy metals such as cadmium accumulation in corn plant tissue and increase corn growth in cadmium contaminated soil [14].Biofilm growth on PDB+NB media has a lag phase in the first 4 hours of incubation during the initial growth period, then an log phase occurs at the 4 th hour of observation until the end of the log phase occurs at the 52 nd hour of observation.Biofilm then experienced a static phase from the 52 nd to the 80 th hour, then began to enter the death phase at the 84 th hour until the end of the observation.

Biofilm Growth Curve
Biofilm is known to be used as a bioremediation agent for heavy metals such as Cr (VI), to determine the Cr (VI) reducing activity, contaminant administration is carried out at the peak of the log phase to see its effect on cell growth, because if Cr (VI) is administered at the beginning of growth it can result in the microbes in the biofilm are unable to grow [11].In general, microbial growth is divided into 4 phases: the lag phase (adaptation), log phase (exponential), stationary phase and death phase.Biofilms on the PDB+NB combined media experience a lag (adaptation) phase for 4 hours and continue to increase for 48 hours, then begin to enter a static and death phase.Cell growth increases because cells can adapt to growth conditions in the new environment and will slow down when resources become scarce until they stop when resources run out [15].From the growth curve, it was found that the biofilm had reached the peak phase at the 52 nd hour of observation, so Cr (VI) could be added to the media.).
Biofilms were added with Cr (VI) contaminant after reaching the peak of the log phase (approximately 52 hours) and their growth was observed every 6 hours.Biofilms when grown on Cr (VI) contaminated media are able to tolerate toxicity and can grow at levels of 5 and 50 mg L -1 (Figure 2).Biofilm growth on Cr (VI) contaminated media experiences a rapid adaptation phase less than 6 hours, then log phase for 18 hours and a static phase until the end of observation (48 hours).
Biofilms when grown in aerobic conditions are able to tolerate toxicity and can grow well on media containing 5 or 50 mg L -1 of Cr (VI) (Figure 1).Biofilm growth on Cr (VI) contaminated media accelerates the log phase, namely 18 hours, and experiences a static phase until the end of observation.Cr (VI) contamination in the media affected biofilm growth because isolates respond to heavy metal ion concentrations by activating the expression of several sets of genes, this causes a faster lag phase then decreases after the log phase showing efficient chromate reduction ability, and at the end of the observation isolates experience resistance so that the static phase takes longer [16]- [18].Analysis of the residual levels of Cr (VI) in the supernatant fraction showed that Cr (VI) had been successfully reduced by the biofilm (Table 1).Biofilms treated with Cr (VI) 5 and 50 mg L -1 were able to reduce Cr (VI) to below the threshold value in the first 6 hours with residual levels in the media of 1.93 and 2,15 mg L -1 and continues to decrease with each observation.At the end of the 48 th hour of observation, the biofilm was able to reduce Cr (VI) to a level of 1.19 mg L -1 both in the Cr (VI) 5 and 50 mg L- 1 .
Based on research results, biofilms have reduce Cr (VI) with initial levels of 5 and 50 mg L -1 to below the threshold value in the first 6 hours.Based on Republic of Indonesia Government Regulation number 22 of 2021 concerning the Implementation of Environmental Protection and Management for NAB Cr (VI) in liquid waste of 2.5 mg L -1 [19].Residual levels of Cr (VI) in the biofilm continued to decrease until the end of the observation, but the biofilm was not able to reduce Cr (VI) completely within 48 hours.The decrease in Cr (VI) levels in biofilms can be caused by the constituent components of the biofilm itself which consists of EPS such as proteins, polysaccharides, lipopolysaccharides, lipids and others, where this EPS is able to act as a permeability barrier to strengthen bacterial resistance to toxic heavy metals and show the ability to biosorption in accumulating nutrients and soluble and insoluble metals [1], [20].Previous research shows that the increasing levels of Cr (VI) contaminants in the media will increase the concentration of EPS in the biofilm, EPS production in biofilms can protect and inhibit Cr (VI) from entering cells [21].EPS contains proteins that can be consumed during the Cr (VI) reduction and immobilization, in addition to functional groups in EPS that can be complexed with heavy metals [22].Other research shows that capsular EPS is the main location of copper metal in the reduction process by biofilms [23].The biofilm reduction ability of Cr (VI) treatment of 5 mg L -1 had an ability of 61.45% in the first 6 hours of observation and continued to increase with each observation, until the end of the observation it was recorded that the biofilm had the ability to reduce Cr (VI) 76.19%.While the biofilms treated with Cr (VI) 50 mg L -1 , the capacity was 95.69% in the first 6 hours, and it was recorded that until the end of the observation, the biofilm had an ability to reduce Cr (VI) of 97.62%.
The biofilm in this study was recorded to have a Cr (VI) reduction ability 0f 61.45% and 95.69% in the first 6 hour of observation by the two biofilm treatment.Biofilm with a higher Cr (VI) treatment, namely 50 mg L -1 , has a higher reduction ability, because the higher the Cr (VI) concentration results in higher and increased biofilm growth, as in previous research, biofilm formation was lower in media with Cr (VI) contamination of 25 mg L -1 compared to 50 mg L -1 , namely 48% and 62%, indicating that there is cell adhesion in the biofilm [24].

Conclusion
The biofilm-based biofertilizer which is a combination of functional microbes on the West Slope of Mount Lawu is tolerant to the toxicity of Cr (VI) contamination at levels 5 or 50 mg L -1 .Biofilm can reduce Cr (VI) to below the threshold value of 1.93 and 2.15 mg L -1 in less than 6 hours in both Cr (VI) treatments.Therefore, to create a healty and safe environment and agricultural land from heavy metal pollution, it is necessary to use biofilm as a biofertilizer to replace inorganic fertilizer which also has potential as a bioremediation agent.Further research needs to be done regarding the interaction of biofilms with the plants above them so that they can help plant growth in soil contaminated with the heavy metal Cr(VI).

Acknowledgment
This research is Penelitian Unggul Terapan (PUT-UNS) with contract number 228/UN27.22/PT.01.03/2023 and the author would like to thank Sebelas Maret University, Surakarta for funding this research, as well as Prof.Dr. Ir. M.M.A Retno Rosariastuti, M.Si who has guided throughout the research.

Table 2
Cr (VI) reduction ability by Biofilms.