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Short communication: A competitive exclusion study reveals the emergence of Bacillus subtilis as a predominant constitutive microorganism of a whey reverse osmosis membrane biofilm matrix
Microbial attachment and colonization on separation membranes lead to biofilm formation. Some isolates within the biofilm microflora acquire greater resistance to the chemical cleaning protocols on prolonged use of membranes. It is thus likely that the constitutive microflora might compete with each other and result in certain species emerging as predominant, especially within older biofilms. To understand the microbial interactions within biofilms, the emergence of predominance was studied in the current investigation. An 18-mo-old reverse osmosis membrane was procured from a whey processing plant. The membrane pieces (2.54 × 2.54 cm2) were neutralized by dipping in Letheen broth. The resuscitation step was done in tryptic soy broth (TSB) at 37°C, followed by plating on tryptic soy agar (TSA) to recover the constitutive microflora. Distinct colonies of isolates were further identified using MALDI-TOF as Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, Bacillus subtilis (rpoB sequencing), and 1 unidentified species each of Exiguobacterium and Bacillus. Further, the competitive exclusion study helped establish the emergence of predominance using a co-culturing technique. Fifteen combinations (of 2 isolates each) were prepared from the isolates. Pure cultures of the respective isolates were spiked in a ratio of 1:1 in TSB and incubated at 37°C for 24 h, followed by plating on TSA. The enumerated colonies were distinguished based on colony morphology, Gram staining, and MALDI-TOF to identify the type of the isolate. Plate counts of B. subtilis emerged as predominant with mean log counts of 7.22 ± 0.22 cfu/mL. The predominance of B. subtilis was also validated using the process of natural selection in a multispecies growth environment. In this instance, the TSB culture with overnight-incubated membrane piece (with mixed-species biofilm) at 37°C for 12 h was inoculated in fresh TSB and incubated for the second cycle. Overall, 5 such sequential broth-culture incubation cycles were carried out, followed by pour plating on TSA plates, at the end of each cycle. The isolates obtained were identified using a similar methodology as mentioned above. The fifth subsequent transfer depicted the presence of only 1 B. subtilis isolate on plating, thereby validating its predominance under the conditions of the experiment.
). The multispecies biofilms are known to be more complex and tend to form thicker and more stable biofilms, which could be due to the release of extracellular polymeric substances (EPS) by various bacterial species (
). Even the cleaning and sanitization protocols prove to be ineffective for removing such resilient biofilms. Such biofouling not only results in a reduced flux rate but also serves as a constant source of contamination, hence leading to premature replacement of separation membranes and economic losses faced by the dairy industry (
). Previous studies have reported the presence of Enterococcus, Staphylococcus, Micrococcus, Streptomyces, Corynebacterium, Bacillus, Klebsiella, Aeromonas, Pseudomonas, Escherichia coli, Streptococcus, Chryseobacterium, and bacteria from Firmicutes and Proteobacteria phylum as constitutive microflora found on dairy separation membranes (
Resistance of the constitutive microflora of biofilms formed on whey reverse-osmosis membranes to individual cleaning steps of a typical clean-in-place protocol.
). When competition is focused on a single resource in a common setting, one microbial population outcompetes the other, hence emerging as predominant in a constitutive microflora. Several factors can be responsible for the emergence of predominance, such as competition for the nutrients, faster-growing rate of one microorganism over the others, production of metabolites by the cells, production of bacteriocin, secretion of broad- and narrow-spectrum toxins with coupled privatized antitoxin, and so on (
). The coexistence of all strains depends mainly on 3 factors: the relative growth rate of the microorganisms present in a mixed species, a ratio of the inoculum, and an effective range of toxins (
). Therefore, it is important to understand the emergence of predominance within membrane biofilms. This could provide vital information on the development of mature biofilms on prolonged use of membranes and the potential for selective species to generate resistances leading to the predominance of one microorganism over others within a biofilm matrix. This information would be useful to create targeted clean-in-place (CIP) strategies to eliminate the resistant biofilm microflora and extend the service life of filtration membranes in the dairy industry in addition to reducing cross-contamination.
An 18-mo-old reverse osmosis whey concentration membrane was procured from a whey processing commercial plant located in the Midwest region of the United States. The used membrane was drawn after the completion of the regular CIP cycle. The CIP protocol involved a 5-wash-step process with an intermittent caustic flush at the end of each wash step. A typical cycle included caustic wash, acid wash, enzyme wash, caustic wash 2, followed by a sanitizer wash, per the recommendations of the manufacturer (Table 1). The whey filtration process in that plant was a continuous run used to filter 350 gallons of cheese whey/min at about 15 to 19°C. The routine CIP protocol involved the combination of alkaline, acid, and enzyme wash at the end of each cycle. The membrane was sealed aseptically in a plastic wrap before bringing it to our laboratory for analysis purposes.
Table 1Cleaning cycles with clean-in-place time and temperatures
Resistance of the constitutive microflora of biofilms formed on whey reverse-osmosis membranes to individual cleaning steps of a typical clean-in-place protocol.
with slight modifications. A cross-sectional piece was aseptically cut out from an 18-mo-old membrane with the help of a reciprocating saw (DWE 304, Dewalt Industrial Tool Co., Towson, MD). Aseptic conditions were maintained by sterilizing the blade of the electric saw and wiping the surrounding area with 70% ethanol before cutting the membrane. Six membrane pieces (2.54 × 2.54-cm2) were cut with a pair of sterile scissors, and any chemical residue on the membrane pieces was neutralized using Letheen broth (Puritan ESK sampling kits with pre-filled Letheen broth, Puritan, Guilford, ME). The composition of Letheen broth is designed to support the growth of a variety of microorganisms, while lecithin helps neutralize quaternary ammonium compounds, and the presence of Tween 80 enables it to neutralize the phenolic disinfectants and hexachlorophene present in the disinfectant; together, they neutralize ethanol (
). Three neutralized membrane pieces were resuscitated by suspending each membrane piece in a bottle containing 200 mL of tryptic soy broth (TSB; Bacto, Rockville, MD) and incubating for 12 h at 37°C. It is also a common incubation temperature used for resuscitation of microorganisms with a variable time length. The TSB, being a nonspecific medium, was used for the resuscitation process to provide a suitable environment for a variety of the stressed intact microorganisms to grow (
). After resuscitation, the bottles were taken out from the incubator and the broth was properly mixed by shaking the bottle containing the suspended membrane piece 25 times in a 1-foot arc within 7 s before plating (
). Serial dilutions (up to 10−7) were pour plated on tryptic soy agar (TSA) and incubated at 37°C for 24 h to enumerate the constitutive microflora. The membrane used for separation operated under conditions close to mesophilic growth (the feed for the reverse osmosis membrane was in the range of 15–19°C), which would mostly support the growth of mesophilic organisms in the biofilm matrix. Distinct colonies obtained on the plates prepared via enrichment process were distinguished based on colony morphology and Gram staining. Based on the similarity in colony morphology, multiple colonies were picked up (2–5 of each type of colony morphology) and were sent out for MALDI-TOF identification at the Veterinary Science Department, South Dakota State University, Brookings. The information was consolidated and presented. The MALDI-TOF resulted in the identification of most of the isolates except 3 isolates, which were identified only to the genus level. Out of these, 1 Bacillus isolate, later also identified as the predominant species of the constitutive microflora, was identified up to its species level using rpoB sequencing (Food Science, Cornell University, Ithaca, NY; GenBank accession no. MT650127).
The competitive exclusion study helped establish predominance using the coculture technique. Six isolates were identified based on colony morphology and Gram staining followed by MALDI-TOF identification and were named VQ1, VQ2, VQ3, VQ4, VQ5, and VQ6. The list of the isolates is presented in Figure 1. All isolates were individually grown in TSB at 37°C for 24 h, and the plate count exhibited an average mean count of 7 log cfu/mL. For the competitive exclusion studies, using the coculture technique, 6 isolates were paired into 15 possible combinations of 2 isolates each (Table 2). For each of these combinations, the overnight-grown isolates were suspended in a 1:1 ratio in TSB, and the cocultures were incubated at 37°C for 24 h. Serial dilutions were prepared, and plating was carried out on TSA. As explained before, the isolates on the plates were distinguished and identified. Finally, the plate counts were compared to establish the predominance of one culture over the other in the coculture growth.
Figure 1Identification of constitutive microflora based on colony morphology and MALDI-TOF.
The results of the coculture growth were further validated using the process of natural selection in a multispecies growth environment. For the natural selection study, a 2.54 × 2.54-cm2 piece of 18-mo-old reverse osmosis membrane was suspended in Letheen broth to neutralize any chemical residue, and then the membrane piece was suspended in TSB and incubated at 37°C for 12 h (cycle 1 incubation). The subculturing of the broth was followed by 5 cycles of subsequent transfers (
). Simultaneously, serial dilutions were prepared at the end of each cycle and plated on TSA using the pour plate technique. The enumerated organisms were identified. The flowchart of the experimental design is shown in Figure 2. All the experiments were carried out in duplicate, and the data were calculated for means values and standard deviations. The culturing techniques used to isolate constitutive microflora revealed the presence of gram-positive as well as gram-negative bacteria. The distinct isolates obtained using the enrichment process under the conditions of experimentation were distinguished based on colony morphology and Gram staining. Individually streaked distinct isolates were then outsourced for MALDI-TOF identification. From these, 6 isolates were identified as Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, 1 unidentified species of Exiguobacterium, and 2 species of Bacillus. Out of the unidentified species, 1 Bacillus strain depicting predominant characteristics (explained below in coculture experiment) was outsourced for rpoB sequencing and was identified as Bacillus subtilis. The other 2 isolates, which were identified at the genus level only, Exiguobacterium (VQ2) and Bacillus (VQ4), were different from the species identified from the constitutive microflora of the 18-mo-old membrane biofilm. Although the MALDI-TOF results did not exactly identify the species levels for these microorganisms (VQ2 and VQ4), they were differentiated from the rest of the isolates based on colony morphology and Gram staining. For the ease of working, these microorganisms were assigned isolate codes, which are depicted in Figure 1, along with their colony morphology and species name based on MALDI-TOF identification or rpoB sequencing. A previously validated protocol was used to isolate the biofilm microflora. In our previous studies, we successfully isolated biofilm microflora to recreate in vitro single and multispecies biofilms in our laboratory (
Resistance of the constitutive microflora of biofilms formed on whey reverse-osmosis membranes to individual cleaning steps of a typical clean-in-place protocol.
). A similar type of biofilm constitutive microflora was previously reported on spiral wound membranes, where species associated with Actinobacteria and Bacillus genera were observed (
) on whey reverse osmosis biofilms have reported the presence of Bacillus as part of biofilm microflora but did not report the presence of some other types of microflora found in the current study. This could be due to variations such as feed or operational parameters, as these factors are reported to greatly affect the diversity of bacteria colonizing membranes (
To establish the emergence of predominance within the biofilm microflora of the 18-mo-old membrane used in our study, the interaction between 15 possible cocultures was investigated. All of the isolates were used to prepare combinations. Each coculture combination consisted of 2 isolates and expressed different colony morphology on TSA plates after the coculture interaction. The isolates obtained on the plates were identified using MALDI-TOF. The plate count of different isolates in the cocultures was recorded, and the log10 counts of each coculture combination were compared. Bacillus subtilis (VQ1) expressed itself in all the coculture combinations with a mean log count of 7.22 ± 0.22 cfu/mL (Table 3). In addition, it inhibited the growth of all the isolates in its combinations. It is interesting to note that another isolate, B. licheniformis was expressed equally in a coculture with B. subtilis and was not completely inhibited. From Table 3, it can be observed that for the coculture combinations VQ4 and VQ5, VQ5 and VQ6, and so on, the isolate that emerged as predominant was ultimately inhibited by VQ1 (B. subtilis) when grown under coculture growth conditions. Therefore, it can be inferred that B. subtilis emerged as predominant in a coculture combination growth environment. The data for all coculture combinations are depicted in Table 3. Similarly, previous studies have reported the predominance of spore-forming Bacillus spp. in membrane biofilms (
). This could be associated with commonly present gram-positive microorganisms or thermoresistant spore-forming Bacillus spp. in whey starter population (
A string test was conducted on the colonies of B. subtilis and B. licheniformis isolates to compare the 2 isolates for their competing attributes in terms of EPS. The presence of mucoid colonies has been associated with the predominance of certain strains over others that do not produce mucoid colonies (
). The string test was found to be positive for B. subtilis only, and the string length was observed to be >5 mm (Figure 3), demonstrating the hypermucoviscosity phenotype. This could also be one of the potential contributing factors for the predominance of B. subtilis in a coculture growth. In addition, studies have reported the prolonged use of membranes and the use of specific operational parameters during membrane filtration to be responsible for the emergence of predominance within different bacterial populations in biofilms (
Resistance of the constitutive microflora of biofilms formed on whey reverse-osmosis membranes to individual cleaning steps of a typical clean-in-place protocol.
Resistance of the constitutive microflora of biofilms formed on whey reverse-osmosis membranes to individual cleaning steps of a typical clean-in-place protocol.
reported that selective bacterial species that developed resistance to the chemical cleaning protocols over prolonged use of membrane acquired predominance in the biofilm microflora. A validation study was also conducted to establish the predominance of B. subtilis by natural selection using a nonselective nutrient broth. This experiment allowed the multispecies microflora to interact and demonstrate the emergence of predominance, similarly to that demonstrated in the coculture growth using combinations of 2 isolates each. The first cycle of incubation of membrane pieces in TSB yielded all 6 isolates previously identified as the constitutive microflora of the reverse osmosis membrane. As the cycles of TSB transfers and subsequent incubations progressed, the types of isolates started to decrease, and finally only 1 isolate of B. subtilis was enumerated at the end of the fifth transfer. Therefore, it can be inferred that B. subtilis emerged predominant within both coculture and multispecies growth conditions. Previous studies have reported that the natural selection process in a well-mixed environment accelerates the transition process by recurring life cycles (
). A similar approach was adopted in this study where subsequent transfers were used to continue the process, which resulted in the emergence of the predominance of one isolate over the others. Also, it is important to note that there is a possibility for the isolates in the microbial suspension to coexist by simply maintaining a balance between growth rates and effective toxicity (
Figure 3The string test was found to be positive for Bacillus subtilis, which had a hypermucoid appearance (string size >5 mm) when it formed a string.
Based on the results obtained, it can be concluded that the isolates within the whey reverse osmosis biofilm microflora demonstrated the emergence of the predominance of B. subtilis among all the isolates under the conditions of the experiment. This study thus provides important information related to the constitutive microflora present within an 18-mo-old reverse osmosis membrane and identification of B. subtilis as the predominant species within the biofilm microflora using coculture and mixed-species growth.
ACKNOWLEDGMENTS
This work was funded by Dairy Management Inc. (Rosemont, IL) and supported by the Agricultural Experiment Station at South Dakota State University (Brookings). The authors acknowledge the Veterinary Science Department at South Dakota State University for conducting MALDI-TOF and Cornell University (Ithaca, NY) for conducting rpoB sequencing. The authors have not stated any conflicts of interest.
REFERENCES
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Socially mediated induction and suppression of antibiosis during bacterial coexistence.
Proc. Natl. Acad. Sci. USA.2015; 112 (26216986): 11054-11059
Resistance of the constitutive microflora of biofilms formed on whey reverse-osmosis membranes to individual cleaning steps of a typical clean-in-place protocol.
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