Response surface methodology mediated optimization of Lignin peroxidase from Bacillus mycoides isolated from Simlipal Biosphere Reserve, Odisha, India

Background Lignin is a complex polymer of phenyl propanoid units found in the vascular tissues of the plants as one of lignocellulose materials. Many bacteria secrete enzymes to lyse lignin, which can be essential to ease the production of bioethanol. Current research focused on the study of ligninolytic bacteria capable of producing lignin peroxidase (LiP) which can help in lignin biodegradation and bioethanol production. Ligninolytic bacterial strains were isolated and screened from the soil samples of Simlipal Biosphere Reserve (SBR), Odisha (India), for the determination of their LiP activity. Enzymatic assay and optimization for the LiP activity were performed with the most potent bacterial strain. The strain was identified by morphological, biochemical, and molecular methods. Results In this study, a total of 16 bacteria (Simlipal ligninolytic bacteria [SLB] 1–16) were isolated from forest soils of SBR using minimal salt medium containing lignin. Out of the 16 isolates, 9 isolates showed decolourization of methylene blue dye on LB agar plates. The bacterial isolates such as SLB8, SLB9, and SLB10 were able to decolourize lignin with 15.51%, 16.80%, and 33.02%, respectively. Further enzyme assay was performed using H2O2 as substrate and methylene blue as an indicator for these three bacterial strains in lignin containing minimal salt medium where the isolate SLB10 showed the highest LiP activity (31.711 U/mg). The most potent strain, SLB10, was optimized for enhanced LiP enzyme activity using response surface methodology. In the optimized condition of pH 10.5, temperature 30 °C, H2O2 concentration 0.115 mM, and time 42 h, SLB10 showed a maximum LiP activity of 55.947 U/mg with an increase of 1.76 times from un-optimized condition. Further chemical optimization was performed, and maximum LiP activity as well as significant dye-decolourization efficiency of SLB10 has been found in bacterial growth medium supplemented individually with cellulose, yeast extract, and MnSO4. Most notably, yeast extract and MnSO4-supplemented bacterial culture medium were shown to have even higher percentage of dye decolourization compared to normal basal medium. The bacterial strain SLB10 was identified as Bacillus mycoides according to morphological, biochemical, and molecular (16S rRNA sequencing) characterization and phylogenetic tree analysis. Conclusion Result from the present study revealed the potential of Bacillus mycoides bacterium isolated from the forest soil of SBR in producing LiP enzyme that can be evaluated further for application in lignin biodegradation and bioethanol production. Scaling up of LiP production from this potent bacterial strain could be useful in different industrial applications. Graphical Abstract

ISOLATION AND SCREENING OF PROTEOLYTIC BACTERIA FROM SIDOARJO SHRIMP PASTE AS PROTEASE SOURCE TO EXTRACT THE COLLAGEN FROM MILKFISH SCALES (CHANOS CHANOS)

This research aimed to isolate protease-producing bacteria from Sidoarjo shrimp paste for extracting collagen from milkfish scales. This study began with isolation, followed by screening and purification of protease-producing bacterial isolates. Further confirmation of the isolates’ proteolytic indices and the crude protease production, the enzymes’ efficacy in extracting collagen from milkfish scales were tested, followed by pathogenicity and identification using 16S rRNA molecular technique. The study has successfully isolated 15 proteolytic bacterial isolates using skimmed milk agar, but only isolates of TR-10, TR-4.1.1, and TR-15.1 exhibited prospective proteolytic activity based on their corresponding proteolytic indices of 2.96 ± 0.06, 3.10 ± 0.10, and 3.71 ± 0.48. Although the proteolytic activity of isolates TR-10 (0.22 ± 0.05 U/mL) and TR-15.1 (1.07 ± 0.14 U/mL) was high in a salt medium using peptone as the nitrogen source, only the former showed satisfactory activity to extract soluble collagen from milkfish scales. Based on the 16SrRNA, the TR-10 isolate was identified as Bacillus megaterium. The non-pathogenicity of the TR-10 bacterium signified its promising role as a protease source for the halal collagen extraction from milkfish scales.

Biotransformations of HBCDs by Rhodococcus Strain Stu-38 and Identification of Transformation Products

1, 2, 5, 6, 9, 10-Hexabromocyclododecanes (HBCDs) are new brominated flame retardants causing serious environmental pollution. Dozens of degradative bacteria have been found with capacity to transform HBCDs. In the present study, an aerobic functional bacterium Rhodococcus strain stu-38 was isolated from enriched culture of mangrove sediment using HBCDs as carbon source. This strain could stereoselectively transform HBCDs, the removal rate was α-＞γ-＞β-HBCD in the mineral salt medium, but was β-＞α- and γ-HBCD in the growth medium, and it selectively transformed γ- HBCD in the seawater. Transformation rate of strain stu-38 was lower than other functional strains, however, seven potential debrominated products of HBCDs were identified by using GC-MS. These debrominated products, included dibromocyclododecadiene, bromocyclododecadienol and bromocyclododecatriene were formed through reductive debromination, hydrolytic debromination and dehydrobromination. Overall, Rhodococcus sp. stu-38 diastereoisomer-specifically transformed HBCDs to various debrominated products in the different cultural media, which highlighted the complicated stereoselective biotransformation of HBCDs.

In Vitro Regeneration of The Endangered Cactus Turbincarpus Mombergeri Riha, A Hybrid of T. Laui x T. Pseudopectinatus

Turbinicarpus mombergeri is a cacti species formed by a hybridization process between Turbinicarpus laui and Turbinicarpus pseudopectinatus. Under natural conditions, it is very difficult for two species be genetically compatible for hybridization, and to produce flowers at the same time. Thus, T. mombergeri is a very interesting and a rare species. Unfortunately, the current populations are decreasing and now it is considered critically endangered. The aim of this research was to develop a successful protocol for propagating T. mombergeri using the in vitro culture techniques. Seed disinfection was performed with Plant Preservative Mixture, and 80% of germination occurred at day 45 in Murashige-Skoog medium. The shoots were cut longitudinally, and the segments were transferred to media containing 2.22 or 4.44 µM benzyladenine to induce shooting. The generated shoots were highly hydrated, and presented abundant callus. The hyperhydricity was controlled by reducing salt medium concentration, by increasing calcium levels and by using polyethylenglycol. The reduction of callus was attained by adding tri-iodo benzoic acid. Vigorous and thick shoots were generated in medium containing urea, and rooting improved in the presence of 0.5 µM indoleacetic acid. Plantlets with normal morphology were obtained, and the survival rate of the plants in soil was 80%. The methodology developed represents an alternative for propagation of T. mombergeri under controlled conditions for commercial or conservation purposes.

Low-Density Polyethylene Film Biodegradation Potential by Fungal Species from Thailand

Accumulated plastic waste in the environment is a serious problem that poses an ecological threat. Plastic waste has been reduced by initiating and applying different alternative methods from several perspectives, including fungal treatment. Biodegradation of 30 fungi from Thailand were screened in mineral salt medium agar containing low-density polyethylene (LDPE) films. Diaporthe italiana, Thyrostroma jaczewskii, Collectotrichum fructicola, and Stagonosporopsis citrulli were found to grow significantly by culturing with LDPE film as the only sole carbon source compared to those obtained from Aspergillus niger. These fungi were further cultured in mineral salt medium broth containing LDPE film as the sole carbon source for 90 days. The biodegradation ability of these fungi was evaluated from the amount of CO2 and enzyme production. Different amounts of CO2 were released from D. italiana, T. jaczewskii, C. fructicola, S. citrulli, and A. niger culturing with LDPE film, ranging from 0.45 to 1.45, 0.36 to 1.22, 0.45 to 1.45, 0.33 to 1.26, and 0.37 to 1.27 g/L, respectively. These fungi were able to secrete a large amount of laccase enzyme compared to manganese peroxidase, and lignin peroxidase enzymes detected under the same conditions. The degradation of LDPE films by culturing with these fungi was further determined. LDPE films cultured with D. italiana, T. jaczewskii, C. fructicola, S. citrulli, and A. niger showed weight loss of 43.90%, 46.34%, 48.78%, 45.12%, and 28.78%, respectively. The tensile strength of LDPE films cultured with D. italiana, T. jaczewskii, C. fructicola, S. citrulli, and A. niger also reduced significantly by 1.56, 1.78, 0.43, 1.86, and 3.34 MPa, respectively. The results from Fourier transform infrared spectroscopy (FTIR) reveal an increasing carbonyl index in LDPE films culturing with these fungi, especially C. fructicola. Analysis of LDPE films using scanning electron microscopy (SEM) confirmed the biodegradation by the presence of morphological changes such as cracks, scions, and holes on the surface of the film. The volatile organic compounds (VOCs) emitted from LDPE films cultured with these fungi were analyzed by gas chromatography-mass spectrometry (GC-MS). VOCs such as 1,3-dimethoxy-benzene, 1,3-dimethoxy-5-(1-methylethyl)-benzene, and 1,1-dimethoxy-decane were detected among these fungi. Overall, these fungi have the ability to break down and consume the LDPE film. The fungus C. fructicola is a promising resource for the biodegradation of LDPE which may be further applied in plastic degradation systems based on fungi.

Molecular identification of insecticide degradation by gut bacteria isolated from Helicoverpa armigera of Cotton plants

The cotton bollworm Helicoverpa armigera occurs as a major pest in many economically important crops, including cotton, pigeon pea, chickpea, pea, cowpea, sunflower, tomato, sorghum, pearl millet and other crops. Intestinal microorganisms play important role in the degradation of diet components of insects. In order to know the role of gut bacteria in insecticide resistance five insecticides Chlorpyriphos (20% EC), Cypermethrin (25% EC), Malathion (50% EC), Quinalphos (25% EC), Triazophos (40% EC), were selected for the insecticide degradation studies. All the bacterial isolates from the gut of lab and field populations of H. armigera were identified using 16S rRNA gene-based identification and tested for their growth on minimal salt medium (MSM) along with the selected insecticides. A total of 11 bacterial isolates were tested and among them, isolate CL4 (Rhodococcus sp.) was found to grow on minimal salt medium (MSM) and with chlorpyriphos and isolate CL2 (Enterococcus casseliflavus) was able to grow in MSM with chloropyriphos (C22H19Cl2NO3) and malathion (C10H19O6PS2) and no growth was seen in MSM without insecticide (control). Gas Chromatography analysis of the positive bacterial isolate cultures in MSM showed that the isolate CL4 (Rhodococcus sp.) was able to utilize 43.9% of chlorpyriphos and isolate CL2 (E.casseliflavus) was able to utilize 26% of chlorpyriphos and 57.1% of malathion in MSM broth cultures with comparison with the respective control cultures. Findings of the current work suggested that gut bacteria in the field populations of H. armigera plays a role in insecticide resistance

Evidence for Enhanced Dissipation of Chlorpyrifos in an Agricultural Soil Inoculated with Serratia Rubidaea Strain ABS 10

Important mineralization of 14C-chlorpyrifos was found in a Tunisian soil exposed repeatedly to this insecticide. A bacterial strain able to grow in minimal salt medium (MSM) supplemented with 25 mg L− 1 of chlorpyrifos was isolated from this soil. It was characterized as Serratia rubidaea strain ABS 10 using morphological and biochemical analyses, as well as 16S rRNA sequencing. In liquid culture S. rubidaea stain ABS 10 was able to almost entirely dissipate chlorpyrifos within 48 hours of incubation. Although, S. rubidaea strain ABS 10 was able to grow on MSM supplemented with chlorpyrifos and to dissipate it in liquid culture, it was not able to mineralize 14C-chlorpyrifos. Therefore, one can conclude that the dissipation capability of this bacteria might be attributed to its capacity to adsorb CHL. In both non-sterile and sterile soil inoculated with S. rubidaea strain ABS 10, chlorpyrifos was more rapidly dissipated than in respective controls.

Biodegradation of alicyclic amines by a newly isolated hypersaline tolerant strain Paenarthrobacter sp. TYUT067

Alicyclic amines are widely used in several types of industries, and considerable attention has been devoted to possible environmental pollution by alicyclic amines in hypersaline industrial wastewater. In this study, a new hypersaline tolerant bacterial TYUT067 capable of growing in liquid basal salt medium with cyclohexylamine (CHAM) as the sole carbon source and energy source, was isolated from soil, and discovered with highly efficient CHAM degrading ability. The strain TYUT067 was identified as Paenarthrobacter sp. based on 16S rDNA gene sequence, and its degradation characteristic was examined. The results revealed that the isolated TYUT067 could grow well under pH range of 6.5–10.0, temperature from 20 °C to 30 °C. For degradation of 60 mM of cyclohexylamine, 100% degradation could be finished within 120 h. The TYUT067 could degrade 10 mM CHAM under hypersaline conditions (3–5% NaCl, w/v), revealed the hypersaline tolerance of TYUT067. Different type of amines was also tested with TYUT067, the degradations of >90% were achieved toward several alicyclic amines. The current results suggested that TYUT067 was a potential species could be efficiently used for the degradation of alicyclic amines and might be applicable to a hypersaline wastewater treatment system for the removal of alicyclic amines.