The presence of microorganisms in the production of plastic and determination of their potential biodegradation capacity

Uvod: Številni znanstveniki in druge zainteresirane skupine se že leta trudijo odkriti »neškodljivo« ali razgradljivo plastiko in s tem prispevati pri iskanju rešitev proti prekomernemu kopičenju plastike v okolju. Namen: Namen magistrske naloge je ugotavljati prisotnost in število skupin mikroorganizmov pri predelavi plastičnih mas v različne izdelke pri opazovanih proizvajalcih ter te mikroorganizme identificirati. Prav tako smo želeli pri bakterijskih sevih preveriti njihovo sposobnost razgradnje polistirenske (PS) plastične mase. Metode dela: V predelovalni industriji plastičnih izdelkov smo v različnih fazah predelave na površinah proizvodne linije in končnih izdelkih z brisi ter kontaktno metodo odvzeli vzorce za mikrobiološke preiskave. Prisotnost in število mikroorganizmov na surovini in končnih izdelkih smo ugotavljali tudi z izpirki. V prvem delu magistrske naloge smo iz vzorcev osamili in z biokemijskimi ter spektrofotometrijskimi metodami identificirali posamezne bakterije in glive. V drugem delu pa smo opazovali zmožnost razgradnje polimera z ugotavljanjem prozorne cone razgradnje PS prahu v gojišču okrog poraslih kolonij izbranih bakterijskih sevov in merjenjem izgube mase PS v bakterijski kulturi po enomesečni in dvomesečni inkubaciji. Prisotnost biofilmov in potencialno živost bakterij na PS ploščicah smo opazovali z mikroskopiranjem s SEM in fluorescenčnim mikroskopom. Rezultati: Večjih razlik v številu in vrsti mikroorganizmov med tremi proizvajalci in proizvodnimi linijami nismo ugotovili. Prav tako ni bilo razlik v številu mikroorganizmov med posameznimi vzorčnimi mesti proizvodnih linij. Večinoma smo osamili po Gramu pozitivne bakterije. Od 109 vzorcev smo identificirali 39 % bakterij rodov Bacillus, 34 % Staphylococcus in po 10 % iz rodov Micrococcus, Burkholderia in Moraxella. Prozorno cono razgrajenega PS okrog kolonij na gojišču smo dobili pri 3 sevih vrste Bacillus licheniformis, pri 64 sevih pa smo opazili tudi rast kolonij, kar pomeni, da so bakterije uporabljale polimer kot vir ogljika. Izguba mase PS pod vplivom razgradnje izbranih sevov je bila od 1,5 do 8 % pri temperaturi inkubacije 37 °C in od 2,1 do 14,4 % pri temperaturi inkubacije 55 °C. Tudi pri tem so bili najaktivnejši sevi Bacillus in Micrococcus. Koncentracija bakterijskih celic oziroma intenziteta njihovega razmnoževanja v gojišču nista bili v pozitivni korelaciji z njihovo razgradnjo in zmanjšanjem mase PS polimera. Pri opazovanju PS zrn pod vrstičnim elektronskim mikroskopom (SEM) smo s slikami pridobili prikaz biofilma oziroma pritrjenih celic na PS zrna. Celice so se najpogosteje pritrjevale na hrapavi površini. Nekateri sevi so tudi po 60 dnevih inkubacije v MSM brez vira ogljika na PS ploščicah, pod fluorescenčnim mikroskopom, kazali respiratorno aktivnost. Razprava in zaključek: Tudi v proizvodnji plastičnih izdelkov so lahko prisotne različne bakterije, ki so sposobne preživeti na površinah PS plastične mase in jo tudi razgrajevati. To so v glavnem sevi Staphylococcus, Bacillus, Micrococcus, ki so pogosti tudi v okolju. Zaradi velike porabe plastičnih izdelkov, ki kot makroplastika ali mikroplastika močno obremenjujejo okolje, so v ospredju številne študije, ki iščejo nove biološke pristope za njeno razgradnjo, ki vključujejo mikroorganizme, genski inženiring, encimsko delovanje ipd. V ospredju so tudi trendi za proizvodnjo biološko razgradljive, za okolje manj obremenjujoče plastike.Introduction: Many scientists and other interested groups are trying for years to discover "harmless" or degradable plastics to prevent excessive accumulation of plastics in the environment. Purpose: The purpose of the Master\u27s thesis is to determine the presence and number of groups of microorganisms during the processing of plastics into various products at the observed manufacturers, and to identify these microorganisms. We also wanted to check the bacterial strains\u27 ability to degrade polystyrene (PS) plastic mass. Methods: In the processing industry of plastic products, we took samples for microbiological tests at various stages of processing on the surfaces of the production line and finished products using swabs and the contact method. The presence and number of microorganisms on raw materials and finished products was determined by washings. In the first part of the master\u27s thesis, we isolated individual bacteria and fungi from the samples and identified them using biochemical and spectrophotometric (MALDI-TOF) methods. In the second part, we observed the ability to degrade the polymer by determining the transparent zone of degradation of PS powder in the culture medium around grown colonies of selected bacterial strains and by measuring the loss of mass of PS in the bacterial culture after one and two months of incubation. The presence of biofilms and the potential viability of bacteria on PS tiles were observed by SEM and fluorescence microscopy. Results: We did not find major differences in the number and type of microorganisms between the three manufacturers and production lines. There were also no differences in the number of microorganisms between the individual sample sites of the production lines. We mostly isolated Gram-positive bacteria. Out of 109 samples, we identified 39 % of bacteria from the genera Bacillus, 34 % Staphylococcus and 10 % each from the genera Micrococcus, Burkholderia and Moraxella. A transparent zone of degraded PS around the colonies on the culture medium was obtained in 3 strains of the Bacillus licheniformis species, and colony growth was also observed in 64 strains, which means that the bacteria nevertheless used the polymer as a carbon source. The loss of PS mass under the influence of the decomposition of the selected strains was from 1.5 to 8 % at the incubation temperature of 37 °C and from 2.1 to 14.4 % at the incubation temperature of 55 °C. Here too, the most active strains were Bacillus and Micrococcus. The concentration of bacterial cells or the intensity of their reproduction in the culture medium was not positively correlated with their decomposition and the reduction of the mass of the PS polymer. When observing the PS grains under a scanning electron microscope (SEM), we obtained images showing the biofilm or cells attached to the PS grains. The cells were most often attached to the rough surface. Some strains showed respiratory activity under a fluorescence microscope even after 60 days of incubation in MSM without a carbon source on PS plates. Discussion and conclusion: Different bacteria can also be present in the production of plastic products, which are able to survive on the surfaces of PS plastic mass and also degrade it. These are mainly strains of Staphylococcus, Bacillus, Micrococcus, which are also common in the environment. Due to the large consumption of plastic products, with which we are greatly burden the environment, many studies are looking for new biological approaches for its decomposition. This include microorganisms, genetic engineering, enzyme action, etc. Trends for the production of biodegradable plastics, which are less harmful to the environment, are also in the foreground