The Effects of Paenibacillus Polymyxa E681 on Antifungal and Crack Remediation of Cement Paste
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Curr Microbiol (2014) 69:412–416 DOI 10.1007/s00284-014-0604-x The Effects of Paenibacillus polymyxa E681 on Antifungal and Crack Remediation of Cement Paste Sung-Jin Park • Seung-Hwan Park • Sa-Youl Ghim Received: 17 February 2014 / Accepted: 13 April 2014 / Published online: 14 May 2014 Ó Springer Science+Business Media New York 2014 Abstract This study investigated the antifungal effects of deterioration factor affecting cement materials and there- cement paste containing Paenibacillus polymyxa E681 fore is particularly important to prevent [6]. Acidic against Aspergillus niger, a deleterious fungus commonly metabolites of fungi can lead to neutralization of contam- found in cement buildings and structures. To test the inated cement surfaces and reduced compressive strength antifungal effects, cement paste containing P. polymyxa [12, 13]. Further, the hyphae of fungi can penetrate into the E681 was neutralized by CO2 gas, and the fungal growth cement structure, leading to physical disruption [10]. inhibition was examined according to the clear zone around The current study confirmed that an organic–inorganic the cement specimen. In addition to the antifungal effects antifungal agent was effective against Aspergillus niger, of the cement paste added with bacteria, calcium crystal common in the interiors and exteriors of building [5, 6, 9, precipitation of P. polymyxa E681 was examined by 10, 12]. According to Do et al. 2005, isothiazoline/carba- qualitative and quantitative analyses. The cement paste mate prevents the growth of Aspergillus niger with cement containing P. polymyxa E681 showed strong antifungal mortar containing 5 % isothiazoline/carbamate produced a effects but fusA mutant (deficient in fusaricidin synthesis) clear zone with no fungal growth. On the other hand, zinc showed no antifungal activity. Crack sealing of the cement oxide and ammonium bromide have been used as algaecide paste treated with P. polymyxa E681 was captured by light agents for the prevention of algal growth which is a major microscope showed fungal growth inhibition and crack contaminant of concrete surfaces in humid environments. repairing in cement paste. Enhanced algaecide properties have been demonstrated on concrete surfaces through the addition of these chemicals [4, 6, 9, 11]. Furthermore, various organo-metallic com- Introduction pounds such as zeolite, zeocarbon, silver, copper, and nickel sulfate have been used for fungal growth inhibition Cement is one of the most commonly used building [5, 6, 9, 10]. These inorganic treatments are effective for materials in the world due to its durability and relatively fungal growth inhibition but often have harmful effects on inexpensive price. However, mature cement structures are humans and the surrounding environment [14]. We neutralized by bicarbonate in the air, resulting in the col- hypothesized that the strong antifungal effects of bacteria onization of bacteria and fungi onto or within the building could be applied to the antifungal cement paste, thus we material as it ages. Fungal growth is a major bio- investigated the potential of antifungal effects of Paeni- bacillus polymyxa E681 in cement paste. The addition of microorganisms to cement paste for the S.-J. Park Á S.-Y. Ghim (&) School of Life Sciences, Institute for Microorganisms, stimulation of organic–inorganic biomineralization has Kyungpook National University, Daegu 702-701, South Korea been applied as a novel approach in cement technology e-mail: [email protected] research [8, 11, 14]. This biomaterial is a progressive concept and environmentally harmless in comparison with S.-H. Park Systems Microbiology Research Center, KRIBB, 111 currently used synthetic polymers in the cement technology Gwahangno, Yuseong-gu, Daejeon 305-806, South Korea field [8, 14]. This concept has been applied in order to coat 123 S.-J. Park et al.: The Effects of Paenibacillus polymyxa E681 413 concrete surfaces [1], enhance the durability of cement– then centrifuged at 12,0009g and 4 °C for 15 min. After sand mortars [3, 9], and repair cracks in cement structures. centrifugation, the sample was washed twice with sterile In addition, carbonate precipitation by bacteria could be distilled water (SDW), and the concentration of fungal used as a self-healing agent for improving concrete dura- spores was adjusted to 106 spores/ml using a hemocytom- bility through biomineralization controlled by bacteria on eter (Marienfeld Co., Germany) [12]. cementitious materials [8]. This study investigated the potential use of P. polymyxa Compressive Strength Test of Cement Mortar E681 in the development of novel, multifunctional cement formulations showing antifungal effects and calcium car- These test strains were inoculated in 300 ml of TSB and bonate precipitation. In our results, E681 induced calcium then cultured at 30 °C for 24 h with 160 rpm shaking. Cell carbonate crystals on calcium-rich medium, and artificial pellets were harvested by centrifugation at 8,000 rpm and cracks in cement paste were sealed by calcium carbonate washed twice with SDW for the elimination of TSB con- crystals produced by E681. In addition, this E681 induced stitutions. The same concentrations of bacterial samples coating of calcium aggregates onto the cement paste sur- were then adjusted to an O.D. of 0.8 at 600 nm, where O.D. face, and the compressive strength of cement–sand mortar value means 109 CFU/ml. A mixture consisting of 240 g of containing P. polymyxa E681 was increased. Above all, the cement, 660 g of sand, and 116.4 ml of SDW was used to growth of A. niger, a well-known fungal strain with dele- cast three cement mortars with dimensions of terious effects on cement surfaces, was severely inhibited 50.8 mm 9 50.8 mm 9 50.8 mm [11]. Six cement mor- by the antifungal properties of P. polymyxa E681. tars containing bacteria were then immersed separately in 1 l of B4 liquid medium containing each treatment, fol- lowed by incubation at 30 °C under aerobic conditions for Materials and Methods 28 days. After incubation, specimens were removed from the B4 liquid medium and dried completely at 25 °C, after Bacterial Strains and Culture Media which the compressive strengths of all specimens were measured using a compressive strength machine (Universal The P. polymyxa E681 which was obtained from the Korea Test Machine, Shimadzu Corporation, Kyoto, Japan) [11]. Research Institute of Bioscience and Biotechnology (Daejeon, Korea) was used in this study. The deleterious Artificial Crack Remediation fungal strain A. niger KCTC6906 was purchased from the Korean Biological Resource Center (Daejeon, Korea). P. Bacterial strains were tested for their ability to remediate polymyxa E681 demonstrates strong antifungal activity cracks in cement paste. Cement pastes were made by against A. niger, whereas the fusA mutant strain shows no mixing aliquots of SDW with cement at a 0.4 water/cement antifungal activity [2]. These bacteria were precultured in weight ratio and then drying at 60 °C for 24 h, after which tryptic soy broth (TSB, Becton Dickson, USA) media, cracks were artificially made by applying physical force. followed by incubation at 30 °C for 24 h with 160 rpm Crack sizes were measured by stereo microscopy (109). shaking. Potato dextrose broth (PDA, Becton Dickson, Bacterial samples suspended in 100 ll of liquid B4 med- USA) was used for inoculum preparation of the fungal ium were applied to cracks in the cement pastes, after strain. B4 liquid and solid medium (0.4 % yeast extract, which the samples were incubated at 30 °C for 24 h. This 0.5 % dextrose, and 0.25 % calcium acetate) were used for process was repeated three times within 3 days at 24-hour the determination of microbiologic calcium carbonate intervals. After 5 days, digital images of the pastes were precipitation (MCCP). Escherichia coli K12, non-calcium captured using a Zentech Digicam. carbonate-forming bacteria with no antifungal activity, was used as a negative control. Preparation and Neutralization of Cement Paste Containing Microorganism Preparation of the Fungal Spore Suspension Paenibacillus polymyxa E681 and fusA mutant strain which To examine the antifungal activity of P. polymyxa E681, a did not show antifungal effects were inoculated into 5 ml spore suspension of A. niger was prepared as described of TSB medium and then cultured at 30 °C with 160 rpm below [12, 13]. PDA media inoculated with the fungal shaking for 24 h. After incubation, 5 ml of each sample strain was incubated at 25 °C for 1–2 weeks in order to was inoculated into 500 ml of TSB and then cultured at generate spores. After culturing, 5 ml of 0.1 % (w/w) 30 °C with 160 rpm shaking for 24 h. Cell pellets were Tween 80 was added, and the fungal colonies were collected by centrifugation at 7,000 rpm, followed by scrubbed using a sterile plastic loop. The suspension was resuspension in 20 ml of SDW. The concentration of each 123 414 S.-J. Park et al.: The Effects of Paenibacillus polymyxa E681 strain was adjusted to 109 CFU/ml by the dilution method specificity of crystals is mainly due to differences among [11]. A mixture containing 50 g of cement and 20 ml of bacterial genera, and bacteria-specific calcium crystal bacterial suspension was prepared, after which 10.0 g precipitation can have different effects on the durability of (±0.5 g) of the mixture was poured into 3.5-cm-diameter cement materials [7, 13]. Thus, various MCCP bacteria plastic dish. After 1 day, cement mortar samples were have been reported and characterized to improve the separated from the plastic dish, transferred into a plastic durability of cement materials. chamber (30 9 20 9 10 cm), and incubated at room tem- To examine the effects of P. polymyxa E681 crystal perature with CO2 gas in order to induce fungal growth. formation on strength improvement of cement, cement The neutralized cement paste containing P.