Rubber Degrading Strains of Microtetraspora and Dactylosporangium

16 Latex clearing protein ( lcp ) found in Actinobacterial strains is reportedly critical for the initial 17 oxidative cleavage of poly( cis -1,4-isoprene), the major polymeric unit of rubber. In this 18 study, we screened 940 Actinobacterial strains isolated from various locations in Sarawak on 19 NR latex agar and identified 18 strains from 5 genera that produced clearing zones and 20 contained lcp genes. We report here the first lcp genes from Microtetraspora sp. AC03309 21 ( lcp 1 and lcp 2) and Dactylosporangium sp. AC04546 ( lcp 1, lcp 2, lcp 3), together with their 22 operon structure. Complete 16S rDNA gene sequence revealed that Dactylosporangium sp. 23 AC04546 is 99% identical to Dactylosporangium sucinum RY35-23 whereas 24 Microtetraspora sp. AC03309 is 98% identical to Microtetraspora glauca IFO14761. 25 Morphological changes and the spectrophotometric detection of aldehyde and keto groups in 26 rubber samples incubated with the strains confirm the strains’ ability to degrade rubber-based 27 products.

Sarawak is recognised internationally as one of the world's 25 biological hot spots, with rich biodiversity, distinctive ecosystems, and extraordinary biological elements 1 .The Sarawak Biodiversity Centre (SBC) (https://www.sbc.org.my/) has been investigating and documenting the diversity of species in the State, and since 2005 and have collected more than 13,000 Actinobacteria strains, isolated from 137 various locations across Sarawak.Our interest in novel strains of Actinobacteria stems from studies showing that these bacterial strains degrade a large variety of organic materials, have high catabolic capacity, and are resilient in the face of unfavourable environmental conditions 2 .Moreover, the same species of Actinobacteria growing in different locations or ecological niches are known to produce environmentally specific metabolites 3 .Using microbes such as Actinobacteria that have evolved genes encoding catabolic enzymes functionally capable of degrading modified rubber are a potential solution for end-of-life management of rubber products 4 .In addition to this, most rubber degrading strains discovered to date belongs to the phylum, Actinobacteria and are rarely found among Gram-negative bacteria and fungi 5 .While Sarawak with its wide range of biodiversity therefore provide unique microenvironments that contributes to the novelty of Actinobacterial strains.
There is great interest in developing environmentally sustainable methods for removing rubber wastes globally.Natural rubber (NR) is highly modified through compounding and vulcanisation in industrial processes and is used in more than 50,000 products today 6,7 .
Natural and synthetic isoprene rubber, primarily composed of highly unsaturated hydrocarbon poly(cis-1,4-isoprene), are widely used.Difficulties in reusing rubber materials due to their resistance to thermal and chemical degradation (REF) results in majority of them being discarded in landfills.Robust, economical, and easily scalable methods for degrading rubber products into minimally hazardous compounds that ideally can be reused are badly needed.
Rubber degrading enzymes, known as rubber oxygenases, produced by microbes are a key discovery for biological rubber degradation.There are 2 groups of rubber degrading bacteria: (i) strains that produce clear zones on NR latex agar and (ii) strains that require direct contact with rubber substrates for subsequent degradation [8][9][10] .In both cases, enzymes produced by the bacteria extracellularly cleave rubber polymers to mixtures of low molecular products (C20, C25, C30, and higher oligo-isoprenoids as end products) that can be taken up by the bacteria 11 .
Our present survey of the SBC's collection of Actinobacteria is geared towards discovering novel rubber degrading Actinobacteria as degraded products obtained from NR or synthetic polyisoprene can differ in their molecular weight, and number of isoprene units, depending on the bacterial strain used 12 .We are thus interested in characterizing the latex clearing protein (lcp) genes in strains that can degrade rubber, as these may have evolved differently in each genus and modulated the strains' functional activity 13 .Little is known about the breakdown mechanism, microorganisms and enzymes involved in rubber degradation.To augment existing information and evaluate the diverse activities in these group of bacteria, the discovery and characterization of new rubber degraders and lcp genes is necessary.

• Screening for NR Latex Degrading Bacteria
To identify rubber degrading strains from SBC's Microbial Natural Product Library (MNPL), random miniaturised screening using 6-well plates for bacteria producing clear zones on NR latex agar were conducted.Eighteen (18) strains were found capable of degrading NR latex, producing ~1 to 2 mm clearance surrounding the colony.By standardizing the clear zone size and day of clear zone detection, we were able to compare the activity of the strains (Fig. 1).• Identification of NR Latex Degrading Bacteria All strains producing clear zones were successfully identified using through partial 16S rDNA gene having blast homology ranging from 99 to 100% (MT005091, MT005089, MT005088, MT005098, MT005101, MT005095, MT005096, MT005104, MT005094, MT005105, MT005103, MT005100, MT005099, MT005102, MT005097, MT005093, MT005090, MT005092).Blast homology of 98.7 -99.5% are putative novel species, 99.6 -99.8% are putative known species and 99.9% and above are identical or closely related species 14 .
Clear zone forming strains were distributed among 5 genera: Microtetraspora sp.rRNA gene suggests that they are not identical (Fig. 2). Figure 2. Rubber degrading strains 16S rDNA evolutionary relationships of taxa together with top 1 blast homology in NCBI database.The evolutionary history was inferred using the Neighbor-Joining method conducted in MEGA X.
• Profiling of Lcp Genes in NR Latex Degrading Bacteria Lcp genes (498 to 1099 bp) were amplified from the NR latex degrading bacteria found in our screen (MN148090, MT241322, MN148093, MN148092, MN148094, MN148095, MT252675, MN148097 MN148098, MT241320, MN148096, MT241323, MT241319, MT241318, MT241317, MT252676, MT241321, MN148089).The lcp genes blast homology was conducted in UniProt database and gave results showing that homology of the selected strains ranged from 81.2% to 100% identity.Lcp gene for both genera has not been recorded in the NCBI database, therefore we choose to further characterize these strains through: (i) morphological studies (Plate 1), (ii) their ability to utilize rubber products as the sole carbon source, and (ii) identification of genes related to rubber degradation through genome sequencing.For strain AC03309, lcp1, lcp2, oxiA and oxiB genes are organized in the same transcriptional unit and orientation (Fig. 3a), while for strain AC04546, lcp 1 was not located in the same transcriptional unit (Fig. 3b).Similar observation was seen in other rubber degrading containing 3 lcp genes, Streptomyces sp.strain CFMR7 and Actinoplanes sp.strain OR16 15,16   • ATR-FTIR Analysis of Degraded Rubber Materials ATR-FTIR spectroscopy is a useful tool to determine the formation or disappearance of functional groups of materials that indicate degradation of the original material (Fig. 4).

Microtetraspora
During rubber utilization, lcp catalyzes the oxidative C-C cleavage of poly(cis-1,4-isoprene) in NR as well as in synthetic rubber by the addition of oxygen (O2) to the double bonds, leading to a mixture of oligonucleotide-isoprenoids with terminal keto and aldehyde groups (endo-type cleavage) 11,17 .The cleavage products are of different lengths, ranging from C20 (four isoprene units) to higher oligo-isoprenoids 17,18 .

Discussions
We were able to quickly screen a portion of this collection by modifying the screening method using NR latex agar in 6-well plates instead of latex agar overlay technique as described by Braaz et al., (2004).We also prolonged the incubation period from 1 week to 4 weeks as some Actinobacteria strains are known to be slow growers.Using 6-well plates, we prepared NR latex agar (without overlay) in smaller volume, utilizing minimal space and less NR substrate.Separation of media by wells also avoid cross contamination especially by sporulating strains.Similar method using NR latex agar in 24-well plate was not successful in detecting the formation of clear zone.Using MSM broth added with NR latex in test tubes, 6well plate and 24-well plate did not show changes in the turbidity of the medium.Based on the screening results, the size of clearing zone on NR latex agar differs within each genus, suggesting each species may have evolve their ability to degrade rubber differently (Fig. 1).
Lcp genes of clear zone forming strains in this study were compared to lcp genes available in the NCBI database (Table 2).Majority of lcp genes deposited in the NCBI database were from Streptomyces sp., but the closest blast homology to Streptomyces sp.However, no further studies on these strains have been reported to the best of our knowledge.
Comparison between 16S rDNA gene of AC04546 and DSM 43158 showed 98% similarity, however no sequence was deposited for DSM 44333.Lcp genes from Microtetraspora sp.AC03309 and Dactylosporangium sp.AC04546 also did not cluster together when compared to other biochemically characterized lcp (Fig. 5), their amino acid sequences were also separate (21.4% to 94.1% similarity) from other known lcp genes (Table 1).Almost all rubber-degrading Actinobacteria, including Streptomyces, Nocardia, and Rhodococcus species have a single lcp homolog 15 .We believe that Actinobacterial strains adapt by incorporating lcp genes into their chromosome through their plasmid (G.polyisoprenivorans VH2) which leads to the presence of more than 1 unique lcp homolog as seen in Microtetraspora sp.AC03309 and Dactylosporangium sp.AC04546.Previous reports indicate that different lcp produces a variety rubber degraded products (molecular weight, number of isoprene units, functional groups, etc) 12 .This would most likely be due to the different or synergistic mechanism of lcp, which is yet to be explored.
Most of the clear zone producing strains (61%) were isolated from soil sample collected from Kiding Village forest area (6 separate soil samples).Kiding village settlement was founded in the 1840s and is located 1,300 m above sea level and is only accessible by a 3-hour hike.While all the other NR latex degrading strains were isolated from secondary forest soil samples.The isolation sites for all the strains have no obvious rubber wastes or rubber materials present, so there does not appear to be an apparent evolutionary pressure for the appearance of lcp genes.Nonetheless, these strains do degrade rubber, and it is possible that this function may have been triggered by the presence of rubber particles dispersed in the environment.Recent studies have shown that rubber particles from sources such as tyres are widely dispersed.Sieber et al., (2020), estimated that 218 ktons rubber particles from tyres are mainly deposited on road-side soils (74%), surface water (22%) and in soils (4%).
Alternatively, the presence of latex producing plants in the vicinity may have contributed to the development of lcp in these strains.Further studies would be required to confirm the reasons for this, but this observation highlights the importance of surveying microorganisms from diverse ecosystems (e.g.marine, or fresh water bodies) for their genomic background and functional ability for biodegradation of rubber or other pollutants.
The influence of the local environment on the development of rubber degrading genera is an intriguing question.The locations where the strains were collected are not obviously rubber dense (e.g.rubber plantations, factories or waste sites), but yet homologous lcp genes were found in the genome of the strains, and they are functionally capable of using latex or rubber as a nutrient source.It would be interesting to survey the collection locations for the presence of rubber pollution in the form of microparticles or other sources of latex in the vicinity.Correlative studies of this nature would help elucidate whether lcp genes are solely expressed based on the presence of rubber/latex in the organisms' local environment.
The variation in lcp structure, function and between different genera and species implies that enzyme product may be evolved to break down specific compounds.This observation raises the intriguing prospect that strategic combinations of different enzymes from different lcps may work synergistically in the degradation of latex or rubber.
As can be seen in the rubber utilization studies, minimal differences were observed for tyre samples, similar case was observed after incubating tyre samples for up to 9 months 22 .Unlike other rubber products, tyres are made from vulcanized rubber and ~30% carbon black for reinforcement which makes them more resistant to degradation.Since tyre is the second largest contributor to microplastic pollution in the ocean, we need to improvise preliminary screening methods for tyre degradation by microbes 23 .
NR latex concentrate: Freshly tapped crude latex was washed with 0.002 % Tween 80 by centrifugation (5 min at 19,320 × g); the washing step was repeated 3x.The top layer was used to prepare the latex agar.Equal amounts of 0.002 % Tween 80 were added into the concentrated purified latex, heat sterilized and stored at 4 °C as purified NR latex concentrate for further use.MSM agar with purified NR latex concentrate were then transferred into 6well plates (4 mL per well).Cultures cultivated in liquid broth for 7 to 14 days were spotted (~ 40 µL) onto each agar well.The plates were incubated at 30 °C for up to 4 weeks.
Colonies that produced translucent clearing zones, indicating the degradation of the NR latex, were recorded.

• Identification of NR latex Degrading Bacteria
Strains producing clear zones were identified based on morphological features and molecular techniques.Morphology was observed on ISP2 agar, and sporulation on soil extract agar 25 .Sporulation patterns were observed directly using a 50 long distance (Olympus LMPLFLN; Olympus, Tokyo, Japan).Spore chain structures and colonial morphology were recorded and photo-documented.Molecular identification was made based on the amplification of the 16S rDNA gene using primer 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (59-TACGGYTACCTTGTTACGACTT-39) with the following parameters: 5 min at 96 °C, 30 cycles of 45 sec at 96 °C, 2 min at 55 °C, 4 min at 72 °C and the final extension for 7 min at 72 °C.Amplified products were purified (GFX PCR DNA GE Healthcare) and sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kits based on Sanger's dideoxy sequencing method.
• Genomic DNA Isolation, Nucleotide Sequencing and Sequence Analysis Strains producing clear zones on latex agar were subsequently cultivated in 20 mL ISP2 broth (Shirling & Gottlieb, 1966) in a 250 mL flask at 28 °C, 200 rpm for three to ten days.
Genomic DNA (gDNA) was extracted and purified using method from Moore et al., (2008).
The DNA quantity and quality were verified by spectrophotometric means (Eppendorf Biospectrophotometer basic).The purified DNA samples were subsequently sent for genomic sequencing (Microtetraspora sp.AC03309 and Dactylosporangium sp.AC04546) using Illumina MiSeq by service provider BioEasy Sdn.Bhd..

• Utilization of Rubber Materials
To study rubber utilization on synthetic rubber; samples of rubber gloves and tyres were used as the sole carbon and energy source, fresh latex was included as control.Fresh latex was harvested from 5-year-old rubber trees at a rubber plantation site at Kulim, Kedah.
The latex was brought back and left to solidify at room temperature.Solid latex pieces were then cut into 1 cm x 3 cm latex pieces.Steel free tyre granules (1 to 3 mm) were obtained from a tyre recycling factory (Gcycle Tyre Recycling) in Kedah.Latex gloves (PRO-CARE), disposable and non-powdered, were used in these studies.To remove antimicrobial substances from the latex glove and tyre granules prior to incubation with Actinobacteria, the material was treated with chloroform as follows: 1 g of sample was extracted with 100 mL chloroform for 12 h 30 .
Pre-culture of actively growing strains were cultivated in ISP2 broth, the culture (1 mL) was then transferred into 250 mL test flasks containing 50 mL MSM and 0.5% (w/v) sole carbon source (fresh latex, samples of gloves or tyres) 30 .The inoculated flasks were incubated at 30°C, 180 rpm for 30 days.Test flasks without culture were used as control.All sample studies were carried out in triplicate.

Sample preparation for Scanning Electron Microscope (SEM) and Attenuated Total
Reflection-Fourier transform infrared (ATR-FTIR) were carried out by rinsing the inoculated rubber materials with distilled water, then immersing them in 96% ethanol for 1 hour before air-drying at ambient temperatures For SEM viewing, samples were prepared using the hexamethyldisilazane (HDMS) method.Dried samples were mounted onto a SEM stub, coated with gold using the Quorum Q150T S (Quorum Technologies Ltd) sputter coater (15 min) and viewed using the SEM Quanta FEG 650 (Thermo Fisher Scientific).SEM images of Actinobacteria strains were also directly viewed using Field Emission Scanning Electron Microscope (FESEM), Quattro Thermo Fisher Scientific carried out at Curtin Biovalley Sdn.Bhd. in Miri.Strains cultivated on ISP2 agar were cut (1 cm x 1 cm) and directly viewed under the FESEM.
To determine the formation of new, or disappearance of functional groups in the polymer units of the samples, post-incubation samples (fresh latex, latex glove, tyre) and noninoculated samples were analysed using FT-IR Spectrum 400 (Perkim Elmer), equipped with ATR at more than 90% pressure ranging from 4000 cm -1 to 650 cm -1 .The angle of incidence was set at 45 using a ZnSe crystal with 20 active internal reflections.Four scans were coadded with resolution set at 4 cm -1 .

Conclusions
Miniaturised random screening of the SBC's MNPL led to the successful identification of 18 NR latex degrading Actinobacteria.Two strains, Microtetraspora sp.AC03309 and Dactylosporangium sp.AC04546 were explored.Both having more than 1 lcp genes on their chromosome and their operon structure is similar to other reports of functional rubber degrading Actinobacteria.Both strains were able to colonise and degrade rubberbased materials within 30 days of incubation.The discovery of novel lcp genes and confirmation of the strain's biodegradation activity in this study further indicate several important directions to explore to better understand the potential of using Actinobacteria in biodegradation processes.Continuing to collect, screen, catalogue, characterize genomically and confirm the activity of Actinobacteria from diverse environments will help us identify the fundamental environmental or biological factors that result in the ability to degrade rubber or latex.In turn this framework will help guide the development of bio-processes that can be used to degrade rubber products on an industrial scale.Rubber degrading strains 16S rDNA evolutionary relationships of taxa together with top 1 blast homology in NCBI database.The evolutionary history was inferred using the Neighbor-Joining method conducted in MEGA X.

Supplementary Files
This is a list of supplementary les associated with this preprint.Click to download. SupplementaryFigures.pdf

Figure 1 .
Figure 1.Size of clearing zone (mm) on NR latex agar in 6-well plate in relation to the day of clear zone formation for 18 rubber degrading Actinobacteria detected in this study.

•Figure 3 .
Figure 3.The organization and transcription of the lcp gene clusters in strain (a) Microtetraspora sp.AC03309 (b) Dactylosporangium sp.AC04546.Open arrows indicate the genes.Location (bp) are indicated above/ below the genes.

Figure 4 .
Figure 4. Cis-1,4-polyisoprene biodegradation pathway by oxygen attack at the double bond (modified from Linos et al., 2000) AC04842 and Streptomyces sp.AC00383 did not contain any lcp gene (Fig. 2).Streptomyces sp.AC04842 has a larger clearing zone compared to Streptomyces sp.AC00383.The genomic content and functionality of lcp genes in Streptomyces sp.AC04842 will be reported in a following study.Back in 1997, a publication indicated that Microtetraspora sp.strain 3880-19B (DSM 44333) isolated from soil sample collected in Malaysia, and Dactylosporangium thailandense DSM 43158 isolated from Thailand produced clearzone on NR latex overlay agar 20 .

Figures Figure 1
Figures

Figure 3 The
Figure 3

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Strain of interest: Microtetraspora sp.AC03309 (JCM 34240) and Dactylosporangium sp.AC04546 has yellowish orange surface and reverse on ISP2 agar with moderate aerial mycelia.Colony is wrinkled with regular shape.It has oblong shaped sporangia (~1.5 µm in length) with smooth surface, emerged directly from vegetative mycelium, arranged singly or in clusters.It is a mesophilic strain, growing well at 28 ˚C and up to 45 ˚C on ISP2 agar.
Using pairwise distance (MegaX), the amino acid sequence identities among biochemically characterized lcps from Actinobacteria are summarized in Table1.Lcp1 and lcp3 from Dactylosporangium sp.AC04546 showed highest similarity (94.1% and 72.3% respectively) to Lcp2 (plasmid) of G. polyisoprenivorans VH2, while lcp2 from . TATR gene is located next to the lcp genes for both Microtetraspora sp.AC03309 and Dactylosporangium sp.AC04546.LCP1 LCP 2 OxiA OxiB 26,929 -25,649 bp 27,206 -28,435 bp 23,345 -25,645 bp 22, 728 -23,051 bp 28,623 -29,279 bp OxiB 43,562 -44,770 bp 44,838 -46,061 bp 46,070 -48,352 bp 48,217 -48,825 bp TATR 364,201 -364,713 bp 364,865 -366,085 bp 42,790 -43,491 bp a b growing by Day 7 while Dactylosporangium sp.AC04546 was by Day 10.SEM images for rubber materials after 30 days of inoculation showed that the strain was able to grow and utilize fresh latex, latex glove, or tyre as the sole carbon source.Biodegradation of the rubber polymer begins with microbial attachment on the surface (Plate 2b to 2c, black arrow) in comparison to non-inoculated samples (Plate 2a).Once attached, the microorganism releases degrading enzymes through its mycelia, initiating the first step of rubber degradation.This can be seen through the presence of rough and cracked surfaces (white arrow) on the rubber materials (Plate 2b -2c).