24th International Colloquium Tribology - January 2024 215 The Importance of Inocula for Biodegradation Testing of Lubricants Dr. Peter Lohmann 1* 1 Hermann Bantleon GmbH, Ulm, Germany * Corresponding author: plohmann@bantleon.de 1. Introduction In nature, degradation processes of organic materials such as hydrocarbons are strongly influenced by environmental conditions. Here, for example, the temperature, the circulation in waters, but also the presence of bacteria capable of degradation play a major role. Higher temperatures accelerate the degradation process, while in colder regions, biodegradation processes take place only very slowly or will completely stop. Similarly, a sea rich in oxygen due to large tidal range, such as in Brittany, accelerates biodegradation processes [1]. There are several hundred species of petroleum-degrading microorganisms, on land, in freshwater and in saltwater [2, 3, 4, 5]. These microorganisms require special enzymes to convert the hydrocarbons into digestible fatty acids [6]. Short hydrocarbons degrade quickly, longer chains take more time, and complex molecules can take months or years [7]. In addition, most microorganisms cannot metabolize all hydrocarbons in the same way. Rather, they have a „main degradation profile“ (Table 1, [8]). Table 1. Petroleum hydrocarbon-degrading bacteria and their preferred degradation substrates. 2. Relevance for degradation tests Well, what does it matter whether a lubricant has been degraded by 50, 60 or 70% after 28 days in a laboratory degradation test? Even if a lubricant has jumped the 60% hurdle of a test in the laboratory, the speed of real degradation in nature remains completely unclear. For this reason, the focus of the laboratory test is only on the comparison of different lubricants. To determine this comparison with possible advantages in terms of degradation rate for a product, the test parameters must be precisely defined and as comparable as possible. In addition to the general laboratory parameters (GLP), the inoculum with its microorganisms plays the most important role in the degradation process. But can an inoculum, which consequently comes from different sources depending on the location, do justice to this comparability? Even the composition of the microorganisms of an activated sludge from one and the same wastewater treatment plant shows great temporary differences (Table 2, [9]). Table 2. Variability of the „same“ activated sludge. Date Number of diff. genera March 2022 215 June 2022 323 July 2022 235 August 2022 309 216 24th International Colloquium Tribology - January 2024 The Importance of Inocula for Biodegradation Testing of Lubricants The idea of a precise inoculum with a defined set of oil-degrading specialists has already been extensively discussed and rejected. Since then, laboratories mostly used activated sludge from the local sewage treatment plant as an inoculum, as required by various degradation standards. As a criterion for the suitability of the inoculum, only the number of colony-forming units (CFU) is determined. The number of CFU should be in the range of 10 3 - 10 6 / ml colony-forming units in the test vessel. The species or genera of microorganisms are not further questioned, although a close correlation between diversity of degrading microorganisms and biodegradability is evident (Figure 1). Figure 1.: Biodegradation of mixed bacterial strains. 3. Conclusion & Outlook As the „main player“, the inoculum is of enormous importance for testing the biodegradability of lubricants. Due to the large variability in diversity of inocula from wastewater treatment plants, the inoculum should be characterized and go through a process of suitability-check before use. The method of next-generation sequencing (NGS), which can provide information about the individual composition and diversity of the inoculum, seems to be suitable for this purpose. References [1] www.wissenschaft.de/ allgemein/ wie-die-natur-dasoelverdaut (2012). [2] Koska et al. (2011) Hydrocarbon-degrading bacteria and the bacterial community response in gulf of Mexico beach sands impacted by the deepwater horizon oil spill. Appl. Environ. Microbiol., 77(22), 7962-74. [3] Liu, Z. and Liu, J. (2019) Evaluating bacterial community structures in oil collected from the sea surface and sediment in the northern Gulf of Mexico after the Deepwater Horizon oil spill. MicrobiologyOpen, 2. [4] Looper, J. K. et al. (2013) Microbial comunity analysis of Deepwater Horizon oil-spill impacted sites along the Gulf coast using functional and phylogenetic markers. Environ. Sci: Processes Impacts, 15 (11), 2068−2079. [5] Leahy, J. G., and R. R. Colwell. (1990) Microbial degradation of hydrocarbons in the environment. Microbiol. Rev. 54: 305-315. [6] Maeng, J. H. et al. (1996) Diversity of dioxygenases that catalyze the first step of oxidation of long-chain n-alkanes in Acinetobacter sp. M-1. FEMS Microbiol. Lett. 141: 177-182. [7] Rehm, H. J., and I. Reiff. (1981) Mechanisms and occurrence of microbial oxidation of long-chain alkanes, p. 175-215. In A. Fiechter (ed.), Advances in biochemical engineering, vol. 19. Springer-Verlag, Berlin, Germany. [8] Xu, X. et al. (2018) Petroleum Hydrocarbon-Degrading Bacteria for the Remediation of Oil Pollution Under Aerobic Conditions: A Perspective Analysis. Front. Microbiol. 9: 2885. [9] Weyandt, R. SGS Fresenius, UNITI Workshop Berlin, March 28 th 2023.