Abstract
Filamentous yeast species belonging to the closely related Saprochaete clavata and Magnusiomyces spicifer were recently found to dominate biofilm communities on the retentate and permeate surface of Reverse Osmosis (RO) membranes used in a whey water treatment system after CIP (Cleaning-In-Place). Microscopy revealed that the two filamentous yeast species can cover extensive areas due to their large cell size and long hyphae formation. Representative strains from these species were here further characterized and displayed similar physiological and biochemical characteristics. Both strains tested were able to grow in twice RO-filtrated permeate water and metabolize the urea present. Little is known about the survival characteristics of these strains. Here, their tolerance toward heat (60, 70, and 80°C) and Ultraviolet light (UV-C) treatment at 255 nm using UV-LED was assessed as well as their ability to form biofilm and withstand cleaning associated stress. According to the heat tolerance experiments, the D60°C of S. clavata and M. spicifer is 16.37 min and 7.24 min, respectively, while a reduction of 3.5 to >4.5 log (CFU/mL) was ensured within 5 min at 70°C. UV-C light at a dose level 10 mJ/cm2 had little effect, while doses of 40 mJ/cm2 and upward ensured a ≥4log reduction in a static laboratory scale set-up. The biofilm forming potential of one filamentous yeast and one budding yeast, Sporopachydermia lactativora, both isolated from the same biofilm, was compared in assays employing flat-bottomed polystyrene microwells and peg lids, respectively. In these systems, employing both nutrient rich as well as nutrient poor media, only the filamentous yeast was able to create biofilm. However, on RO membrane coupons in static systems, both the budding yeast and a filamentous yeast were capable of forming single strain biofilms and when these coupons were exposed to different simulations of CIP treatments both the filamentous and budding yeast survived these. The dominance of these yeasts in some filter systems tested, their capacity to adhere and their tolerance toward relevant stresses as demonstrated here, suggest that these slow growing yeasts are well suited to initiate microbial biofouling on surfaces in low nutrient environments.
Highlights
The filamentous yeast strains belonging to S. clavata and M. spicifer and the budding yeast strain belonging to S. lactativora, investigated here, are representative strains from the total number of yeast species previously isolated from a Reverse Osmosis (RO) membrane filtration line for water reuse in a dairy industry (Stoica et al, 2018; Vitzilaiou et al, 2019)
On Malt Extract Agar (MEA) agar, after 12 days incubation at 25◦C, SC colonies were circular with a 2–7 mm diameter, glassy, tough, hirsute, white, convex, and filiform with 1–4 mm mycelium length, while M. spicifer (MS) colonies were circular with a 1–3 mm diameter, butyrous, glistening, soft, whitish, convex, and filiform with 1 mm mycelium
It has already been established that they may cover large areas and still go relatively unnoticed unless selective media are used for their detection, since they are often present in lower numbers and grow slower than bacteria, causing them to be out-grown on non-selective media (Stoica et al, 2018; Vitzilaiou et al, 2019)
Summary
Filamentous yeasts are emerging as highly potent biofilmforming microorganisms in water distribution systems (Doggett, 2000; Babicet al., 2017), residential dishwashers (Zalar et al, 2011; Dögen et al, 2013; Gümral et al, 2016; Zupancicet al., 2016) and in food industrial equipment (Tang et al, 2009; Tarifa et al, 2013; Stoica et al, 2018; Vitzilaiou et al, 2019) These findings indicate that filamentous yeasts can disperse efficiently, attach strongly to different surfaces and create robust hyphal networks capable of surviving several stresses. Biofouling may become an issue for final product quality, if microbial cells from the biofilms are capable of proliferating further down the processing line or in the water permeate during storage, since the water may be used for cleaning or for direct/indirect product contact processes (Casani and Knøchel, 2002; Casani et al, 2005)
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