Biofilm forming microorganisms on various substrata from greenhouse of Botanical Garden “Jevremovac”

Зборник Матице српске за природне науке / Matica Srpska J. Nat. Sci. Novi Sad, № 132, 57—67, 2017 UDC 579.26:061(497.11 Beograd) https://doi.org/10.2298/ZMSPN1732057U N i k o l a D . U N K O V I Ć *1, S l a đ a n a S . P O P O V I Ć 2, M i l o š Č . S T U P A R 1, E l e n a D . S A M O L O V 1, G o r d a n a V. S U B A K O V - S I M I Ć 1, M i l i c a V. L J A L J E V I Ć - G R B I Ć 1, Jelena B. V UKOJEVIĆ1 1 University of Belgrade, Faculty of Biology Institute of Botany and Botanical Garden “Jevremovac” Takovska 43, Belgrade 11000, Serbia 2 University of Belgrade, Institute of Chemistry, Tehnology and Metallurgy, Department of Ekology and Technoeconomics Karnegijeva 4, Belgrade 11000, Serbia BIOFILM FORMING MICROORGANISMS ON VARIOUS SUBSTRATA FROM GREENHOUSE OF BOTANICAL GARDEN “JEVREMOVAC” ABSTRACT: Diversity of subaerial biofilm forming cyanobacteria, algae and fungi was investigated on 10 different substrata from greenhouse of Botanical Garden “Jevremovac”. Out of 37 documented taxa, 16 cyanobacterial and 10 algal taxa were identified. Remaining 11 taxa belong to the Kingdom of Fungi. The highest diversity of biofilm forming microorganisms, a total of 24 taxa, was detected on the corroded metal surface, while significantly lower number of taxa was recorded on other examined substrata. Cyanobacterium Porphy rosiphon sp., diatom Achnanthes sp. and green algae Chlorella sp. and Chlorococcum minu tum were the most frequently encountered photosynthetic components of biofilms. In all analyzed samples, Trichoderma sp., followed by Cladosporium sp. and Rhizopus stolonifer, were the most frequently identified fungi. KEYWORDS: algae, biofilm, cyanobacteria, fungi, greenhouse INTRODUCTION In natural conditions, phototrophic and heterotrophic microorganisms are able to colonize and subsequently form ubiquitous, self-sufficient, miniature microbial ecosystems on all substrata where direct contact with the atmosphere and solar radiation occurs (Gorbushina, 2007). Process of establishing these complex microbial communities, known as subaerial biofilms (SABs), depends on * Corresponding author. E-mail: 57 substratum bioreceptivity, biology of colonizing microorganisms and wide range of environmental factors such as temperature, humidity, pH, solar radiation, water and nutrient availability (Gu and Mitchell, 2006; Macedo et al., 2009). In early stages of SAB formation, cyanobacteria and algae use CO2 from the atmosphere and sunlight as their carbon and energy source and produce metabolites which serve as nutrient source for incoming heterotrophic bacteria and fungi (Ljaljević Grbić et al., 2010). In addition, various air-borne organic and inorganic deposits and animal remains help fill nutrient requirements for heterotrophic SAB forming microorganisms (Suihko et al., 2007). Although most biofilms only contain complex consortia of algae, cyanobacteria, heterotrophic bacteria, fungi and protozoa, in advance stages of the colonisation, more complex organisms such as lichens, mosses and vascular plants can occur (Ljaljević Grbić et al., 2009; Stupar et al., 2014). Changes in the structure and appearance of the substratum occur as a result of biofilm development. Discoloration, due to pigment excretion, depends on specific physiology of the SAB involved species and is influenced by changes in physiological state of the cells and the environmental conditions (Cappitelli et al., 2008; Warscheid and Braams, 2000). Additionaly, SAB forming microorganisms secrete various extracellular polymeric substances (EPS) to maintain moisture levels, enable mutual binding of microbial cells and adhesion to the substratum (Warscheid and Braams, 2000; Macedo et al., 2009). However, EPS may potentially cause alteration of physico-chemical properties of the substrata due to retained water (Brehm et al., 2005; Keshari and Adhikary, 2013). Fungi produced organic acids and enzymes interact with released CO2 resulting in pH change of the substratum, which further facilitates the mechanical degradation (Gorbushina et al., 2007). Moreover, chemical reactions between organic acids and minerals ensue bio-weathering and formation of secondary minerals on the attacked substrata. The aim of this research was to study the diversity of SAB forming microorganisms on different substrata from greenhouse of Botanical Garden “Jevremovac”. MATERIALS AND METHODS Sampling was done in 2010 from different substrata within the greenhouse of Botanical Garden “Jevremovac”, University of Belgrade, Faculty of Biology, Institute of Botany. Sampling site Sampling of SAB forming cyanobacteria, algae and fungi was conducted on various substrata from the greenhouse of Botanical Garden “Jevremovac”. The Botanical Garden was founded in 1874 by the decree of the Ministry of Education of the Kingdom of Serbia, at the suggestion of famous Serbian botanist Josif Pančić. The greenhouse, from which samples were taken, was built in 1892 and covers the area of 500 m2. Since the time of its construction, numerous tropical, sub-tropical 58 and desert plants have been grown in two wings connected by a central dome. From 1892 to 2010 no work was done on the reconstruction of the greenhouse. Today, due to its exceptional architectural value, it is protected by the law. Sampling Samples for algological and mycological analyses were collected from the surfaces of 10 different substrata with visible SAB formation: wood (Wd), stone (St), sand (Sd), clay (Cl), mortar (Mr), concrete (Co), metal (Mt), nylon (Ny), putty (Pt), and glass (Gl) (Figure 1). Figure 1. Examined substrata, with visible alterations, from the greenhouse of Botanical Garden “Jevremovac”: a. wood; b. stone; c. mortar; d. clay; e. sand; f. concrete; g. putty; h. metal; i. glass; j. nylon. 59 Algological analyses Algological analyses were conducted on samples acquired using two methods: scraping and non-aggressive adhesive tape sampling (Gaylarde and Gaylarde, 1998). After rehydration in modified Knöps medium, samples were analyzed using stereomicroscope (Zeiss Stemi DV4) and a light microscope (Zeiss Axio-Imager M1, with software AxioVision Release 4.6). The observed cyanobacteria and algae were identified to species or genus level, on the base of cellular morphology, using appropriate literature (Starmach, 1972; Krammer and Lange-Bertalot, 1988; Komarek and Anagnostidis, 1998; Komarek and Anagnostidis, 2005). Mycological analyses Sampling for the mycological analyses was done using sterile cotton swabs and adhesive tape method. Sterile swab samples were diluted in 10 mL sterile distilled water and shaken mechanically for 10 min, after which 1 mL of the resulting suspensions was inoculated on malt extract agar (MEA) medium with 500 mg streptomycin per liter (Booth, 1971). The inoculated plates were incubated in a thermostat at 25 ± 2 °C. After incubation period of 7 days, pure fungal cultures were obtained by re-isolation of primary isolates onto the selective nutrient med (...truncated)