CHAPTER 4: AIRBORNE ENDOSPORE BIOBURDEN AS AN INDICATOR OF SPACECRAFT CLEANLINESS SPACECRAFT CLEANLINESS
4.5 Discussion
4.4.6 Comparison of biofilm-forming environmental-strain B. subtilis and lab-strain B.
subtilis endospores
The ability to form fruiting bodies could be passed on over 3 generations with intermittent sporulation, germination and outgrowth. Cultures of bacterial endospores (B.
subtilis 3160) were grown on TSA bacterial growth medium. Two weeks after the culture was initiated, to allow time for the formation of bacterial endospores on the surface of the biofilm, an SAS air sampler was used to measure the concentration of spores which could easily be transported by moderate air flow over the surface of the biofilm. It was determined that approximately 100 endospores could be recovered from 1000 L of air passed over the biofilm. A strain of B. subtilis that form biofilm and delicate aerial structure was isolated from Mojave Desert (Figure 4.7). Two weeks after colony formation on TSA, approximately 1000 endospores were recovered when 1000 L of air passed over the culture. This finding demonstrated that under laboratory conditions, surface microbial biofilm can release bacterial endospores that can be transported through the air, resulting in the airborne spread of potentially dangerous microorganisms from surfaces. No significant differences were observed in the unit DPA content and germination properties between these endospores.
spacecraft enclosed habitat, capable to detect as low as 100 endospores L-1 of air when 500 L of air were sampled in 15 min. Apart from technological development, one must fully understand the correlation between AEB level with the total biomass in an enclosed system with no outside ventilation in order to help promulgation of exposure limits and safety levels, as well as maximum allowable concentrations for airborne bacterial spores.
Suspended particulate matter of a diameter less than 10 µm (PM10) was used as a measure of air quality in this study. It was well establish that sustained exposure to elevated PM10 values was associated with a variety of health problems25. Regulatory limits for PM10 values have therefore been imposed26,27. In this study, we have employed the PM10 value, log transformed, as the variable of interest.
In this study, we have measured AEB and total biomass in a closed system under laboratory controlled conditions and everyday indoor environemnts. A quantitative correlation and mathematical model has been established under ideal conditions, such as volume, air flow, turbulence and the number of microbes. The principal components of each AEB and surface bacterial abundance is subjected to the analysis of variance (two- way ANOVA) followed by a pairwise multiple comparisons. Principal component analysis of the pairwise AEB and total biomass in indoor closed environments, such as laboratory, offices, laminar flow hoods, produced two factors that accounts for 49%
(eigenvalue = 3.5) and 19% (eigenvalue = 1.5) of the total variance in the data set.
Clustering of component scores indicates an apparent tendency to form separate cluster assignments under different environments. It is noticed the ratio of AEB and total biomass can be projected with high accuracy using our model system when minimal human activities are involved. As human activities and environmental factors play a bigger role in the system, a positive correlation can still be observed between AEB and
total biomass, but these systems are difficult to model due to increased complexities as shown in separate studies. A more comprehensive study on the effect of PM10 values due to temporal and spatial behaviors is expected in future studies.
We used a three-pronged approach to testify the hypothesis. First, we demonstrated a reproducible bioefficiency among three air samplers (SAS, MEM, E- BAM) to validate the use of SAS in subsequent studies because of its compactness and portability. Second, we performed air and surface sampling inside an AtmosBag under controlled conditions. A two-compartmental mathematical model has been developed to characterize the transport of airborne and surface endospores in this 12-L enclosure. The model was extended to analyze a number of indoor areas, including laminar flow hoods, laboratory and office spaces. Third, we studied the effect of human activities and environmental factors in the correlation of airborne and total biomass in a system.
We describe the system as a two compartment system such that there is exchange of materials between them. q1 and q2 are defined as state variables describing the number of airborne endospores and the number of endospores on the surface. Exchange occur by endospores transgressing some physical barrier, characterized by two rate constants, k12, is the number of endospores transferred from air to surface per unit time and k21
represents the number of endospores aerosolized from surface into the air per unit time.
The basic assumption for the compartmental model is that endospores are distributed homogenously in the entire system. The 2-compartment model structure is given as followed. The model dynamics with state variables can be described in Figure 4.8, where u1 and u2 in this system are delta function inputs. The two rate constants can be measured by fallout plates (n = 10). The number of airborne endospore depositing on surfaces was
20.1 ± 7.2 cfu h-1 and the number of endospores being aerosolized was 2.0 ± 1.4 cfu h-1. Therefore, k12 and k21 were calculated to be 0.0193 cfu min-1 cm-2 and 0.4109 cfu min-1 cm-2, respectively after unit conversion. Solving the system of differential equations, we have obtained plots very close to actual results under ideal situation. While this is a very simplistic model assuming well-controlled condition (especially air turbulence), it truly reflects the environment inside a spacecraft. We then showed qualitatively that the number of total endospores in a closed system was positively correlated with the degree of human activity in Marshall Space Flight Center. Moreover the time course in a spacecraft relevant environment (i.e., the MSFC simulated cabin) of airborne bacterial spores shows a trend that is highly correlated to the time course of the environment, human commensal, and fungal spore concentrations. This implies that a change in airborne bacterial spore concentration can be used to indicate a corresponding change in total microbial content in the cabin.
Air sampling has been carried out in the Atacama Desert to verify the model that we have developed. The results high depend on the environmental factor, such as wind, relative humidity and UV irradiation. PDMS was used as collection substrate for fallout plates. During the day, the air was devoid of culturable microorganisms and fallout plate also indicated zero counts. At night, counts were observed in both air sampling and fallout plates. This shows that there exists an exchange of airborne and surface microbes in the absence of UV irradiation. In addition, the wind speed was 5 times greater than that in the daytime, reaching a peak velocity of 25 km h-1, which may be another reason for the presence of aerosolization and deposition of microbes via wind as an agent. In conclusion, in an outdoor environment, lots of other factors are dominating the airborne
bacterial concentration. In addition, surface bacteria are difficult to extract due to (1) extreme radiation and temperature of the surface, (2) complex soil matrix and (3) sources of contaminations from human and animal activities.
The MEM monitors the concentration of airborne bacterial spores in cabin air as an indicator for total microbial concentration. Spore-forming bacteria of the genus Bacillus are frequently among the most abundant genera in aerobic biofilms, and are likewise among the most abundant genera found in the air and on surfaces. As a biofilm grows, the population of the ever-present sporeforming bacteria and their endospores increase proportionally. In fact, recent reports demonstrate that spore-forming species in environmental biofilms form fruiting bodies, filled with bacterial spores that rise above the biofilm to facilitate their release into the air. Thus, the airborne bacterial spore concentration can be correlated to total microbial concentrations. In this experiment, we also examined the aerosolization properties of a biofilm-forming endospore-forming species isolated from the Mojave Desert. Although it has been reported that properties of endospores depend largely on the sporulation condition, this environmental species still retained its biofilm colony morphology after 3 generations. As expected, these biofilm- forming endospores were 5 times more susceptible to aerosolization than laboratory strain B. atrophaeus endospores that do not form biofilms. Nevertheless, the DPA content, hydrophobicity and germination characteristics of this environmental strain are all similar to B. atrophaeus endospores.