Go green with biotechnology

Recent New Zealand Leather and Shoe Research Association (LASRA) findings are guiding the application of biotechnology to help the New Zealand leather industry develop environmentally sustainable leather processes. Using 16S rRNA gene sequencing, we have isolated and identified a number of indigenous bacteria from the leather industry environment that are being adopted to develop benign leather-processing technologies. We isolated and identified several Bacillus strains from a biofilter used in a leather-manufacturing plant, which exhibited sulphide oxidation activity that are being applied in bioremediation of volatile organosulphur compounds emitted by leather products.

We also discovered a strain of Stenotrophomonas spp with significant and beneficial proteolytic activity in a tannery sludge. The identified strain not only displays collagenase activity but also the ability to reduce hexavalent chromium to trivalent chromium, making it an ideal candidate for biodegradation of tanned waste. Recently, we revisited the natural autolytic processes of degradation of untreated pelts to guide a natural depilation method without any need for additional chemical treatment. In controlled experiments the wool could be removed completely from follicle after two days without obvious damage, and leathers could be processed with mechanical properties, comparable with conventionally processed counterparts. The alkaline protease activity of the isolated bacteria is responsible for the observed natural unhairing.

Production that provides

Leather production not only serves social needs by utilising a meat by-product, but also contributes significantly to the global economy through trade and employment. But the social image of the leather industry is becoming increasingly negative.

In line with New Zealand’s global reputation of being an eco-friendly country, its leather industry has committed to improving its environmental performance and image by adopting sustainable practice throughout the production process and providing high-quality leather products with reduced environmental impact during their life cycle.

Chemicals extensively used during leather production have also been regarded as a source of unpleasant odour sometimes emitted from leather products, especially in cabin upholstery. In certain regional markets, such as Asia, consumer concerns about the indoor or in-cabin air quality relate odour emission with health threats, which impacts their purchase decisions on leather products.

Besides consumers’ preference, the odour has been proved to be caused mainly by volatile organic compounds (VOCs), which are harmful at high concentrations. Concerns for indoor air quality and consumers’ health have driven the implementation of legal regulations applicable to the emission limits of VOCs from products, including leathers, which are becoming increasingly strict. Therefore, the development of leathers with low VOC emissions will not only ensure improved market acceptance, but also contribute to a healthier indoor environment.

Biotechnology such as enzymatic processing has been considered a realistic substitute for current leather production practice. Proteolytic enzymes including alkaline protease and keratinase have been investigated for their potential to replace the large quantities of chemicals used in the unhairing step, which is the most polluting operation.

Since the raw materials used and waste generated during leather production are generally proteinous, the leather industry environment could serve as an ideal resource for the discovery of microbes producing useful proteolytic enzymes. Guiding the application of sustainable biotechnologies to assist the transition of the New Zealand leather industry, LASRA has developed the capability to identify potentially valuable microorganisms from the local industrial environment including storage, sludge and compost, for instance.

The identification of bacterial species is carried out routinely at LASRA by 16S rRNA gene sequencing, which has been widely applied to study bacterial phylogeny and taxonomy, as this conservative gene is believed to be the most common housekeeping genetic marker in almost all bacteria.

LASRA is also establishing platforms to characterise and produce useful microbial enzymes, which could be applied in alternative leather processes. While cleaner enzymatic processes are still being developed, issues related to VOCs emitted from leather products can be addressed by deploying VOC-consuming bacteria during conventional leather production.

As the oldest biological method for removal of undesirable gaseous compounds from air, biofiltration uses microorganisms as the engine of the biotreatment process. Because biofilters operate as fixed-bed bioreactors with immobilised active microorganisms, it is reasonable to hypothesize that VOC-metabolising bacteria can be isolated from biofilters dealing constantly with such pollutants such as those used by tanneries.

In this study, bacteria with the potential to benefit sustainable leather production were isolated from the New Zealand leather industry environment. Isolated strains were identified using 16S rRNA gene sequencing, and a preliminary investigation of the impact of bacterial activity on leather performance was also carried out.

Details of the study

Bacteria with odour mitigation potential were isolated from the soil bed of a biofilter used by a New Zealand tannery. A sample of the soil was mixed with phosphate-buffered saline (PBS) solution to prepare a 10% (w/v) suspension. Serial dilutions of the soil suspension were plated onto LB agar plates, which were then incubated at 37°C. Colonies with visually distinguishable morphology were selected for further studies. Bacteria with proteolytic potential were isolated from sludge and sheep skins. Sludge samples collected from LASRA’s tannery waste treatment plant were suspended in PBS to prepare 10% (w/v) suspensions.

Sheep skins were freshly provided by a local slaughterhouse and cut into halves along the backbone. The right halves were depilated conventionally as a control, and the left halves were kept at ambient temperature until the wool could be manually removed. A piece of skin sample was then taken from each left half and a bacterial suspension was prepared by placing each skin sample in a 50ml centrifuge tube containing 20ml PBS solution shaken at room temperature for four hours at 200rpm.

$25 million
New Zealand exports in footwear last year, ranking 88th in the world. 

55%
Leather footwear amounts to over half of all footwear exported from New Zealand.
World Footwear

Serial dilutions of the bacterial suspensions were plated onto LB agar plates with 2.5% (w/v) skimmed milk at 37°C. The proteolytic activity was detected by the formation of translucent halos around the individual colonies, which was a result of the hydrolysis of casein in the milk.

Identification of the isolated bacterial strains was carried out by 16S rRNA gene sequencing. The candidate colonies were picked up and cultured in 5ml LB broth medium for 24 hours at 37°C. Genome DNA from each culture was extracted and 16S rRNA genes were amplified by PCR using primer pair 27F and 1427R. DNA electrophoresis with 2% agarose gel was employed to verify the successful amplification of the 16S rRNA genes from all the candidate colonies.

The PCR products were purified before being submitted to Massey Genome Service for sequencing. The sequencing results were analysed using the Targeted Loci Nucleotide BLAST and the phylogenetic trees were constructed using the Neighbour-Joining method with P-distance.

Sheep skins were then processed into crust leathers following LASRA’s standard protocol. Physical properties examined included tear strength, tensile strength and percentage elongation at break and grain crack resistance. Comparison was made using four samples from each half of the skin and each group consisted of three skins.

Process and facilities

The biofilter facility used by a New Zealand leather manufacturer was found to be supportive of the growth of plants within it. The air emitted from the production hall after filtration though the soil bed did not provoke any noticeable perception of unpleasant odour. From the soil sample collected in the biofilter, four Bacillus species were identified as potential candidates responsible for the removal of odorous compounds during filtration of the gaseous waste emitted by the leather manufacturer.

These bacteria have been reported to promote plant growth mainly through nitrogen fixation, which was consistent with the overall observation of the biofilter facility where healthy plants were thriving across the entire soil bed of the biofilter. These species have previously been characterised as being interactive with sulphur and ash from coal, indicating their potential application in the bioremediation of leather odour by metabolising the sulphur-containing volatile compounds emitted from leather products.

In other research at LASRA, the volatile compounds emitted by New Zealand leather products have been profiled to identify the odorous molecules, serving as targets to be mitigated by bioremediation. The metabolism of the identified compounds by the Bacillus species isolated in this study has been examined to reveal the responsible bioremediation mechanisms and results are pending. Comparison between the volatile profile of leather products before and after bacterial treatment will demonstrate the efficacy of the proposed strategy for leather odour mitigation. Additionally, the identified species strongly inhibit the growth of pathogenic fungi across a wide range of host plants. Therefore, in addition to reducing unpleasant leather odours, the identified stains might be applied to develop novel biocontrol methods, to prevent damage to leather caused by fungi.

Consistent with LASRA’s previous finding on the proteolytic potential of tannery sludge, the present study isolated and identified the responsible bacterial strain from the sludge treatment. From 16S rRNA gene sequencing and phylogenetic analysis, the dominant strain in the tannery sludge exhibited 99% sequence identity with a Stenotrophomonas strain.

Members of the genus Stenotrophomonas have been reported to have keratinase activity and also to reduce hexavalent chromium to benign trivalent chromium. Our preliminary experiments have revealed that the identified strain exhibits collagenase activity as well as tolerance to hexavalent chromium. These results suggest that this strain might usefully contribute to the biodegradation of tanned leather waste.

Currently, we are optimising the enzyme production conditions and the bioremediation of hexavalent chromium. The activity of enzymes produced by the identified strain, including collagenase, keratinase, lipase, neutral protease and alkaline protease are currently being characterised. The application in beamhouse operations of proteases produced by the identified strain is also being investigated with promising progress.

x10
Air pollutants that generate that ‘new car smell’ have been found at levels up to 10 times regulatory limits inside some models.
New Scientist

What was found

The natural wool loosening induced by microbial protease has attracted interest for a long time. In this study, it was found that after storage for 48 hours, the wool throughout the skin could be removed by pulling effortlessly, leaving empty follicles and a slightly damaged grain. Proteolytic bacteria on the skins, which might be responsible for the observed natural wool loosening were identified as Aeromonas spp, Proteus spp and Wohlfahrtiimonas spp. Aeromonas spp has long been known to produce extracellular proteolytic enzymes. Proteus species secrete protease as one of the virulence factors associated with the infection process and disease.

Among strains of Proteus spp, one identified in this study has previously been found to be present as one of the bacterial strains causing wool loosening. Wohlfahrtiimonas spp has been demonstrated to be associated with myiasis, infection with the larvae of parasitic flies. The strong chitinase activity of Wohlfahrtiimonas spp may play a role in the metamorphosis of the fly.

Lucilia sericata larvae linked to the infection of Wohlfahrtiimonas spp are used as an alternative treatment for recalcitrant and chronic wounds, which could be attributed to the various peptidases within the excretion secretions. While the effect of chitinases on the depilation of sheepskin is still inconclusive, the proteolytic enzymes from maggots might contribute to the natural unhairing process.

In a subsequent study, the protease activity and the substrate specificity of the enzymes secreted by the identified strains have been determined. The potential enzyme candidate can then be produced by fermentation and applied, experimentally, and replace lime and sodium sulphide in the depilation process. The methods developed using sheepskin as a model can also be adopted on other materials such as cow hides.

The crust leathers obtained from conventional chemical depilation and natural wool loosening were examined for their physical properties. Leathers produced from natural processing presented comparable strength properties to their chemically processed counterparts, such as tear strength, tensile strength, elongation. It is noteworthy that the grain crack resistance of the leathers processed with natural wool loosening is significantly higher than that of the leathers processed traditionally. The improvement in the physical characteristics of leather processed from skins with natural wool loosening might be attributed to improved uptake of tanning chemicals, which has been observed in enzymatically processed leather.

Bio-upgrading

Our research is aimed at enabling the New Zealand leather industry to produce high-quality leather products with a much-reduced environmental footprint. The application of biotechnology helps the leather industry adopt a more sustainable practice. Our results demonstrate that the VOC profile of leather products can be improved by treatment with bacteria isolated from a biofilter. The bacteria isolated from sludge treatment presented protease activity, which is being investigated for biodegradation of leather waste as well as enzymatic depilation of hide and skin.

Extracellular protease-producing bacteria were isolated from locally sourced sheep skins. The crust leathers processed from skins influenced by those bacteria presented comparable or even improved physical properties, compared with their conventionally processed counterparts.

JY Liu and G Holmes spoke in Dreseden, Germany, at the IULTCS Conference. A full paper is available on request.