Antibacterial and Anti–Biofilm Activities of Acetone Extracts of Usnea sp. against Mixed Cultures of Bacteria from Soak Liquor Samples and Tank Surfaces

Long-term or improper use of antibacterial agents utilized in the soaking process has led to the resistance of some bacteria in the leather industry. New agents may be the solution to combat these antibacterial resistant bacteria in the soaking process. As a natural resource, lichens are known to have many biological activities. In previous studies, we demonstrated that the acetone extracts of several lichen species including Usnea sp. may have potential antibacterial and anti–biofilm properties against some Bacillus species, which were isolated from different soak liquor samples. In the present study, it was questioned whether the same bioactivities of acetone extracts of Usnea sp. can be seen in the mixed cultures of tank surface samples and pre-and main soak liquor samples, which were obtained from different tanneries. Although the extracts did not show noteworthy antibacterial effect against one of the tank surface samples (inhibition ratios; 6.5–16.22 %), inhibition percentages were detected as 69.32 and 46.33 at the concentrations of 240 and 120 µg/mL for the other tank surface sample. The anti-biofilm potential of the extracts was tested on the sample where the antibacterial activity of the extracts was not observed. One of the mixed culture of samples from the tank surface could not be inhibited by the extracts in terms of bacterial growth. However, the extracts were tested on this biofilm-forming sample and detected more than 50% inhibition. Furthermore, the extracts inhibited the growth of the mixed culture of bacteria from pre-soak liquor by the percentages of 78.96, 61.5, 51.3, 45.1, and 33.4 at the concentrations of 240, 120, 60, 30 and 15 µg/mL, respectively. On the other hand, the same antibacterial efficacy could not be observed in the other mixed culture from pre-soak liquor sample obtained from a different tannery whereas this sample formed a biofilm structure. The mixed culture of samples from the main soaking process was inhibited by the extracts at the inhibition percentages of 62.13–78.17 at the concentrations of 240- 30 µg/mL. Similar results were also obtained for the other sample (64.6–76.5%) from main soak liquor sample obtained from a different tannery. In conclusion, lichen extracts may have potential antibacterial and anti-biofilm properties against the mixed culture of bacteria from tank surface, pre-and main soak liquor samples and maybe alternatively utilized in the leather industry.

Antibacterial Potential of Six Lichen Species against Enterococcus durans from Leather Industry : Evaluation of acetone extracts obtained from several lichen species as alternative natural antibacterial agents

Antibacterial resistant bacteria are a significant problem in the hide or skin soaking process due to their destructive properties on finished leather. Lichens may be a solution to overcome this resistance problem. Enterococcus durans (99.86%) was isolated from soak liquor samples. For screening of possible antibacterial effects of lichen acetone extracts, six lichen species (Hypogymnia tubulosa, H. physodes, Evernia divaricata, Pseudevernia furfuracea, Parmelia sulcata and Usnea sp.) were examined by nine-fold dilution against E. durans. H. tubulosa, H. physodes and E. divaricata extracts showed antibacterial effects at the concentrations of 240 μg ml−1, 120 μg ml−1 and 60 μg ml−1 whereas the extracts of P. furfuracea had an antibacterial effect at 240 μg ml−1 and 120 μg ml−1. On the other hand, P. sulcata had no antibacterial effect. The most successful lichen extract was determined to be Usnea sp. at the concentrations of 240 μg ml−1, 120 μg ml−1, 60 μg ml−1, 30 μg ml−1 and 15 μg ml−1. In conclusion, lichen extracts seem to have potential antibacterial efficacies against E. durans.

De-Oiled Karanja Cake as Potential Bio-Additive for Low Salt Raw Skin Preservation

The most commonly practiced preservation processes for raw hide/skin is by application of common salt (NaCl) due to its wide availability, cost effectiveness and bacteriostatic activity. During the soaking process, the salt present in the skin dissolves in water, thereby generating high total dissolved solids (TDS) levels causing pollution to the environment. Hence, an eco–friendly material without salt or with lower quantities of salt is needed for the preservation of the skin. In this present study Karanja de-oiled cake (Millettia/Pongamia pinnata) extract was studied for its antibacterial activity, minimal inhibitory concentration and minimum bactericidal concentration for preservation. The reverse phase high performance liquid chromatography (RP-HPLC) analysis shows the presence of karanjin (0.2%) and pongamol (0.02%) and these compounds may be responsible for the antibacterial activity against skin deteriorating bacteria. The preservation trials with karanja de-oiled cake 15% along with less salt 15% showed no hair slip, no putrefaction, reduced bacterial load to approximately 35%, compared to control skins at 25º-35ºC for 21 days. The preserved skins on rehydration showed that the soak liquor of experimental skins had a considerable reduction in TDS (about 67%) compared with conventional soak liquor. On processing these skins into leather, the experimental skins were found to possess comparable strength properties (tensile, tear strength) and organoleptic properties as conventional salt-based preserved skins. This study concludes that conventional salt-based preservation of skins can be replaced by an eco-friendly preservation method using karanja de-oiled cake along with less salt.

Supercritical Carbon Dioxide Based Skin Preservation: Solving the Soak Liquor Effluent Crisis of the Leather Industry

Salt-based preservation of hides/skins contributes to about 50% total dissolved solids (TDS) in tannery wastewaters. In this study, raw skins have been preserved by exposing them to a continuous flow of supercritical carbon dioxide (SCCO2) in a pressurized reactor. The process was carried out in reactors of two different capacities to ensure scalability. The skins thus dried could be stored at room temperature for a period of 30 days. The SCCO2–dried skins were less conducive for microbial growth than wet-salted skins. The soak liquor of SCCO2–dehydrated skin showed a 90% reduction in chloride content and significantly lower BOD and COD levels than soak liquor from wet-salted skins. The leathers produced from SCCO2-preserved skins and wet-salted skin had no significant quality differences. As SCCO2 systems have been reported as alternatives to all other unit operations, establishing SCCO2–based preservation will complete the circle of total leather manufacture with SCCO2.

Antibacterial Activities of Lichen Derived Extracts against Different Bacillus Species from Soak Liquor Samples

In the leather industry, some bacterial strains may become resistant to antibacterial agents utilized in the soaking process due to long-term use and/or not using in sufficient doses. Alternative approaches or novel agents need to be investigated to overcome antibacterial resistance of bacteria present in the soaking process. These alternative approaches may be from natural resources such as lichens which are known to have various biological activities such as antibacterial, antifungal etc. For this purpose, soak liquor samples from different tanneries were collected and eight isolates from these samples were identified by cultural and molecular techniques. Also, the antibacterial effects of acetone extracts of Hypogymnia physodes, Evernia divaricata, Pseudevernia furfuracea and Usnea sp. at different concentrations were tested on these isolates. They were all Gram (+), rod shaped, oxidase (+), catalase (+), protease (+). Six isolates had lipase activities. The isolates were assigned to Bacillus toyonensis, B. mojavensis, B. subtilis, B. amyloliquefaciens, B. velezensis, B. cereus, and B. licheniformis in molecular analyses. The acetone extracts of Evernia divaricata totally killed B. toyonensis, B. mojavensis, B. amyloliquefaciens, and B. subtilis at the concentrations of 240, 120, 60 and 30 µg/ml, respectively. These extracts had also significant antibacterial efficacies on B. cereus, B. velezensis, B. licheniformis at the concentration of 240 µg/ml. The acetone extracts of P. furfuracea had a great inhibitory effect on the growth of most species (80.24-88.65%) only at the concentration of 240 µg/ml. H. physodes acetone extracts totally killed B. amyloliquefaciens and had considerably high suppressive effect on the growth of other tested bacteria at the concentrations of 120 and 240 µg/ml. Usnea sp. acetone extracts had inhibitory effect on Bacillus species (86.6-97.9%) even at the 30 µg/ml concentration. In this respect, lichens may provide an alternative approach for the leather industry to overcome bacterial resistance to the antibacterial agents.