The overall goal of the following experiment is to isolate soil fungi and bacteria that colonize and degrade biodegradable mulch films. This is achieved by first incubating biodegradable mulch films in mesh bags in the soil long enough to permit substantial colonization by native soil microbes. Following incubation, the mulches are extracted and dilution plated, which yields single isolated colonies of each culturable type of intimately associated microbe.
Then isolated microbes are patched onto fresh surface, dis infested, biodegradable mulches, as well as onto agar plates with or without a carbon source. In order to compare growth, the results are then confirmed in liquid medium. The results show colonization and or degradation by certain isolates based on scanning electron microscopy and can be verified by gel permeation chromatography.
Don't forget that working with unidentified fungal and bacterial isolates can be hazardous, and precautions should always be taken while performing this procedure such as wearing gloves, sealing culture tubes and dishes, and working in a biosafety cabinet. After incubating mulch pieces in soil and preparing media and reagents according to the text protocol, cut new mulch pieces into 4.25 centimeter squares to decontaminate the mulch pieces. Autoclave them if it will not alter or degrade them for non autoclavable mulch pieces such as those made from plastics.
After preparing a biosafety cabinet according to the text protocol, place the mulch pieces in the decontaminated hood in rows and incubate the mulch pieces under UV light for two hours, using a clean sterilized forceps and beginning with the front row and working towards the back to avoid placing hands and arms over the newly decontaminated surfaces. Flip over the mulch pieces, placing the dis infested side down on a clean area, exposed to UV light, but not previously in contact with mulch. After two hours of UV treatment on each side, again, working from front to back.
Use sterile forceps to remove the mulch pieces from the biosafety cabinet and place them into sterile dry covered containers such as foil covered beakers. Store them at room temperature in darkness under a sterile transfer hood. Using aseptic technique, lay the UV dis infested mulch pieces on the surface of agar plates setting down one corner first and gently, letting the remainder of the square roll into contact with the agar.
For non-water permeable films, place four 10 microliter drops of molten semi-solid, minimal medium atop the corners of the mulch pieces to provide a water and nutrient source for initial colonization of hydrophobic plastics. Do not touch the surface of the mulch pieces. With the pipette tip, the drops will harden into beads.
Incubate the covered plates upright overnight at room temperature to solidify. Then store the plates agar side up in the dark at four degrees Celsius. All materials necessary for the bioassays are now prepared and you're ready to begin isolation of potential mulch degrading fungi from mulch pieces that have been buried in the soil.
Dig up mesh bags from the soil and remove mulch pieces from the mesh bags. Use a sterile spatula and forceps to gently remove the excess soil from the mulches without disturbing the soil that is clinging to the surface. Cut BDM film into one centimeter square pieces until 0.5 grams of material is recovered.
For each replicate sample transfer, 0.5 grams of mulch pieces and attached soil into 25 milliliter. Culture tubes containing 9.5 milliliters of PBS add 0.5 grams of soil to the soil only control tubes or to samples where the mulch is completely degraded to break apart any biofilms and detach cells from mulch pieces. Vortex the tubes for 30 seconds at high speed, then sonicate them in a water bath for 10 minutes and vortex them again.
This should yield a homogenous suspension of microbial cells that were associated with the mulch pieces. For a serial dilution of samples. Prepare one culture tube with 4.5 milliliters of sterile PBS and fill three wells of a deep well plate with 450 microliters of PBS per sample from the original tubes of suspension.
Transfer 0.5 milliliters into the 4.5 milliliters of sterile PBS to obtain a 5.0 times 10 to the negative third dilution after vortexing at high speed for 30 seconds, add 50 microliters to 450 microliters of PBS in the 96 well plate mix by pipetting gently up and down 10 times repeat until dilutions of up to 5.0 times 10 to the negative six are complete. Use flame sterilized bent glass rods to evenly spread 100 microliters from each of the dilutions across the agar surface of PDA plates that contain chloramphenicol to inhibit bacterial growth. Store plates upright for 30 minutes to let the liquid soak in.
Then seal the plates with paraform to avoid spores escaping from unidentified isolates. Incubate inverted plates at 20 degrees Celsius in the darkness for five days to allow both quick and slow growing fungi to form colonies in a biosafety cabinet. Examine the dilution plates for single isolated colonies using a single sterile toothpick.
Gently touch a colony and then touch the agar of fungal minimal medium, fungal minimal medium plus plastic and glucose minimal medium plates in that order to inoculate agar beads atop plastic films. Rub the toothpick gently on the surface of the agar dot and then swipe it across the plastic film. Repeat the inoculation from the same source colony until there are four.
Replicate streaks on each plate after incubating the plates at 20 degrees Celsius in the dark for five days. Compare plates to identify isolates that grow on mulch pieces better than they grow on minimal medium containing no added carbon aside from agar. These are potential mulch degraders.
The GMM plate should contain the most growth since it is a positive control plate in which the medium contains glucose. Soil isolates may not be entirely pure at this point, so use a sterile toothpick to scrape inoculum from the fungus, colonizing the mulch and streak it for isolation onto potato dextrose agar, then seal the plate and incubate for five days. After the five day incubation, single isolated colonies will have arisen.
Confirm that these new purified colonies show mulch colonization in the plate bioassay similar to that, observed with the original potentially impure isolates. Once potential mulch degraders have been identified by the plate bioassay verify using a liquid bioassay in this final confirmatory assay, even agar is lacking under the mulch. Pieces are truly the sole carbon source for microbial growth in this stringently prepared medium to begin the assay at a surface dis infested mulch piece to a fungal medium lacking any other added carbon for each replicate of a microbial isolate.
Prepare three tubes for inoculation. One, an experimental tube with five milliliters of minimal medium here labeled FMM containing a mulch piece of the predetermined size. Two, a negative control with five milliliters of minimal medium with no carbon source.
And three, a positive control with five milliliters of glucose containing liquid. Here labeled GMM. Additionally, prepare a replicate tube of minimal medium containing each mulch to be tested, but do not inoculate it.
This control tube will reveal any microbial growth resulting from contamination after growing and collecting fresh spores. According to the text protocol, in a sterile transfer hood inoculate the previously prepared culture tubes with a million spores or yeast cells, seal the caps and incubate in the dark without shaking at 20 degrees Celsius. Observe the samples weekly for growth.
Mycelial growth of fungi on mulch pieces may be visible within the first week after inoculation, especially at the edges of the mulch pieces for planktonic yeast monitor growth by optical density readings at 600 nanometers because filamentous fungi are likely to attach directly to the mulch pieces. Assess the growth by eye and confirm by light microscopy and or scanning electron microscopy in minimal medium only controls. Look for minuscule white flex that are too small to register a change in optical density.
Using spectro photometric methods. Use a paster pipette to aspirate the flex and observe them using differential interference. Contrast microscopy after isolating three potential mulch degrading isolates in the plate bioassay from both a cellulosic mulch called weed guard plus that was our positive control and a starch based plastic mulch called Biore.
They were incubated in the liquid bioassay for 68 days and harvested before subjecting them to scanning electron microscopy. As expected. Fungal hyphy were observed on all of the samples except XX and YY where rod-shaped cells were observed for the isolates on our cellulose control mulch.
No growth was observed in the FMM only controls as shown here in the UN inoculated controls for the weed guard plus mulch residual trachea elements of the plant-derived fibers were only detectable as slight pock marking seen in rows on some of the fibers, though fungal hyphy were observed in all three inoculated samples of our cellulose control mulch. Trachey elements were plainly visible only in the SS samples suggesting that digestion of cellulosic material revealed the lignified trachea elements beneath isolate SS was tentatively identified using 18 s ribosomal, DNA as a soda mye within the order sodas for the starch based plastic mulch. Isolates no significant growth was observed in the minimal medium only controls single white specs visible upon swirling in the culture tubes of isolates, VV and ZZ were observed using differential interference contrast microscopy for isolate vv.
The spec was a spore mass, presumably residual from the initial inoculation. For isolate zz. The mass was composed of loose mesh of hyphy, approximately 0.2 millimeters in diameter, suggesting that the original spore inoculum had germinated but had not grown beyond the mass pictured here.
As seen here in the UN inoculated controls for the starch based plastic mulch, a bumpy texture is observed. The identity of the white features is unknown, but they're absent in a film colonized by isolate VV shown here. Both the white particles and bumps are gone in a film colonized by isolate zz.
In addition, the latter sample showed the uniform occurrence of fissures across the BDM surface isolates. VV and ZZ were tentatively identified using at s ribosomal DNA as Penicillium and ACE within the order of hypo respectively. Though this method can provide insight about biodegradation of plastic mulches for the agricultural industry, it can also be applied to any other plastic whose biodegradation in the environment is in question.
For example, plastics that might end up in the marine environment. Are those used for the food industry? I.
