For the past several years, air and water purification systems have been developed and used. This technology is based on the combined activities of plants and microorganisms as they function in a natural environment. More recently, researchers have begun to address the problems associated with indoor air pollution. Various common houseplants are currently being evaluated for their abilities to reduce concentrations of volatile organic compounds (VOCS) such as formaldehyde and benzene. With development of the Space Exploration Initiative, missions will increase in duration, and problems with resupply necessitates implementation of regenerative technology. Aspects of bioregenerative technology have been included in a habitat known as the BioHome. The ultimate goal is to use this technology in conjunction with physicochemical systems for air and water purification within closed systems. This study continued the risk assessment of bioregenerative technology with emphasis on biological hazards. In an effort to evaluate the risk for human infection, analyses were directed at enumeration of fecal streptococci and enteric viruses with the BioHome waste water treatment system

Ellender, R. D.

Johnson, Anne H.

Watkins, Paul J.

English

NASA Technical Reports Server

N91-24053
NEW RESEARCH ON BIOREGENERATIVE AIR/WATER
PURIFICATION SYSTEMS
Anne H. Johnson
Science and Technology Laboratory
National Aeronautics and Space Administration
John C. Stennis Space Center
Stennis Space Center, MS. 39529-6000
R.D. Ellender
Paul J. Watldns
Department of Biological Sciences
University of Southern Mississippi
Box 5153
Hattiesburg, MS. 39606
INTRODUCTION
For the past several years, the Science and Technology Laboratory at Stennis Space Center (SSC) has
been involved in the development and appfication of air and water purification systems. This technology is
based on the combined activities of plants and microorganisms as they function in a natural environment.
Early efforts dealt with the use of artificial or constructed wetlands for wastewater treatment. Numerous
communities as well as corporations have adopted this technology. In fact, all of the wastewater at SSC is
treated using these types of systems. More recently, researchers have begun to address the problems
associated with indoor air pollution. Various common houseplants are currently being evaluated for their
abilities to reduce concentrations of volatile organic compounds (VOCS) such as formaldehyde and benzene.
With development of the Space Exploration Initiative (SEI), there will be significant increases in mission
duration. Problems with resupply necessitate implementation of regenerative technology. Although the final
system may primarily be based on physicochemical processes, it is feasible to consider the application of
bioregenerative technology for the air/water purification.
Aspects of bioregenerative technology developed at SSC have been included in a prototype habitat known
as the BioHome (Figure 1). A 650 SF structure, the BioHome functions as a pilot system to facilitate analysis
of bioregenerative technology in a semi-closed environment. The ultimate goal is to employ this technology in
conjunction with physicochemical systems for air and water purification within dosed systems.
The BioHome is divided into two regions, one is designated as a living area while the second contains the
wastewater treatment system. This system is a modified version of an artificial wetland, relying on vascular
plants and microorganisms to effect the treatment process. The system is housed within 6 - 8 inch segments of
polyvinylchloride (PVC) pipe ranging in length from i0 to i2.75 ft. and contains plants such as canna lilies
(Ca_a) and bulrush (Scirpus). In addition, there are various types of porous substrate included such as
activated carbon. Due to increased surface area, the substrate material promotes biof'flm development, a
process integral to successful treatment of wastewater (1). In addition, biofilms also play a role in the
presence or absence of bacterial pathogens (2).
Prior to inclusion of bioregenerative air or water purification systems in a closed environment, it is
necessary to fully assess the associated risks. It is expected that wastewater will have a characteristic
microflora, some of which will be pathogenic. Similarly, biological contaminants may be airborne. The bulk
of the latter group will probably originate in the abundant plant material present. There is a potential
problem in closed systems with build-up of airborne microbes that may be attributed to the lack of ozone and
ultraviolet rays. These elements are present outdoors and comprise what is known as the "open air factor" (3).
Consequently, there is a tendency for microbial survival to be enhanced indoors due to the absence of this
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https://ntrs.nasa.gov/search.jsp?R=19910014740 2020-03-19T17:45:23+00:00Z
effect.DevicesuchasHEPAfiltersmaybeusedto reducesomebiologicalairbornecontaminants,however
they will not alleviate the problem. Similarly, chemical contaminants may occur in ambient air. They stem
from a variety of sources including building material, plants, and humans.
Earlier preliminary studies have dealt with partial assessments of biological contaminants in the BioHome.
Data indicated that the wastewater treatment system exhibited tremendous potential for reduction of bacterial
pathogens such as Salmonella (97.53%) and _ (98.52%) (4). Similarly, the biological oxygen demand
(BOD) and fecal coliform counts were significantly reduced (Tables 1, 2). Studies analyzing ambient
microflora revealed relatively low levels of bacteria and fungi present. Bacterial genera included
Escherichia, Flavobaeterium, Klebsiella, Microe0ccus, and Staphyloe.oceus. Fungal isolates were identified as
members of the genera Aspergillus, Mucor, and Penicillium.
The purpose of this study was to continue the risk assessment of bioregenerative technology with emphasis
on biological hazards. In an effort to evaluate the risk for human inflection, analyses were directed at
enumeration of fecal streptococci and enteric viruses within the BioHome wastewater treatment system.
MATERIALS AND METHODS
Fecal Streptococci Analysis: For a period of ten weeks, weekly water samples were taken from both
effluent (segment 1) and effluent (segment 6) sites of the treatment system. Using the membrane fdtration
technique, appropriate volumes of sample were analyzed using Gelman GN-6 0.45 m sterile filters. Following
filtration, the filters were aseptically transferred to KF agar and incubated at 35°C for 48 hours (5). The
density of fecal streptococci/enterococci per 100 ml was caloulated using only those plates with colonies
numbering in the desired range (20 to 60). Verification of isolates was accomplished according to the - _ :
protocols outline in A.P.H.A.'s Standard Methods (5). :
Enteric Virus Analysis: Measured quantities of wastewater were pumped through 90 mm 1MDS Virosorb
membranes for a total of 27 samples. The majority of samples were taken from the effluent sampling port.
Additional samples were obtained from segments 3 and 4 as well as the septic tank. 90 mm membranes were
eluted using 80 ml of 0.1 M glycine, pH 10.5. The eluent from this step was then passed through a 47 mm
Virosorb membrane and e!uted with 5 ml of 0.1 glycine, pH 10.5. Next, 10% PSF and 0.1 (10X) gentamicin
was added, sample pH was adjusted to 7.0, then the sample was incubated at 35°C for one hour. Samples
were then centrifuged at 1900 X g for 20 minutes, filtered (0.20 micron), and distributed into 1.5 ml aliquots
for storage at -70°C. For purposes of inoculation, Linbro plates were prepared from stock MA-104 cells and
allowed to settle for 24 hours, Next, the growth medium (L-15) was removed by aspiration and each
monolayer inoculated with 0.1 ml of undiluted sample. Following an incubation period of one hour at 35°C,
monolayers were covered with 1 ml of maintenance medium and incubation continued. Plates were observed
daily for evidence of cytopathic effect for a total of seven days (6).
RESULTS
Results of the fecal streptococci analysis indicated that the wastewater treatment system significantly
reduced numbers of this group (Table 3). Influent samples over the 10 week period averaged 53 CFUs
(colony forming units)/100 ml. None of the effluent samples exhibited any growth. Consequently, the system
is 100% effective in the reduction of fecal streptococci/enterococci.
To date, no viruses have been isolated from any portion of the wastewater system. 27 samples were
screened for the presence of enterics with no evidence of cytopathic effect.
DISCUSSION
It is encouraging to find that fecal streptococci are _tuaUy removed from the-wastewater. This
group, also known as the Group D streptococci, has been linked to high incidences of urinary tract infections
as well as abdominal lesions and are resistant to numerous antibiotics (7). Similarly, the absence of enteric
108
virusesisapromisingf'mding.Thereareseveralfactorsthatmayaccountfor thelowlevelsand/orabsenceof
thesegroups.First,thesepticityofthetankprecedingtheartificialwetlandmaybesuchthatconditionsare
unfavorablefor bothgroups.FactorsuchashighNI-I3contentmaylimitsurvival,particularlywithrespecto
entericviruses.It isalsopossiblethattherelativenumbersof bothgroupsarecomparativelylowin theraw
wastewater.ThemajorityofsewagethatisusedfortheBioHomestudiesisderivedfromthatwhichis
generatedonsiteatSSC.Consequently,thepresenceorabsenceof aparticulargroupofmicroorganismsis a
reflection of resident microbial population associated with the raw wastewater.
The analysis of these data along with previous studies support the finding that artificial wetlands may
provide a suitable means of reducing the number of pathogens in wastewater (8, 9). Several studies have
documented the advantages of aquatic and wetland plants for the treatment of wastewater (10, 11). It has been
theorized that plants perform two functions in an artificial wetland system. The first is that they provide
increased surface area for microbial attachment; an important consideration since the treatment process relies
on microbial activity. The second function relates to the transport of oxygen to the root zone, or rhizosphere,
thereby producing an aerobic environment (12). The resultant aerobic zone supports a microbial consortium
that effects modification of nutrients, ions, and other compounds while the aerobic/anaerobic interface serves
to enhance the processes of nitrifcation and denitrification (13).
It is interesting to note that plants have additional mechanisms to dictate the types of microorganisms
found within the rhizosphere. Studies by Bowen and Rovira (14) revealed that several regions of the root
produce compounds that leak from the root or may be pumped out as a result of metabolic activity. Such
compounds were identified as inhibitory to certain microorganisms. Broadbent et al (15) theorized that such
antibiotic activity may be involved in significant coliform reductions associated with artificial wetlands.
Similarly, Palmateer et al (16) found that coliform reduction was enhanced substantially during the summer.
This reduction coincided with an anoxic period, suggesting the ability of plants to translocate oxygen to the
rhizosphere, thereby providing an explanation for improved coliform removal in vegetated systems.
These findings are also supported by Seidel (17) whose studies included Juncus effusus, Scirpus lacustris
and Phragmites communis. Seidel maintains that excretions from the plants either partially or completely kill
pathogenic bacteria while heterotrophs are left unharmed. Unfortunately, the author neglected to include
relevant reference material. Consequently, the validity of these finds must be carefully considered.
Pathogens are known to be removed by physical/chemical processes (fdtration and adsorption) and by
biological inactivation and predation (18). However, biot'flm development also plays an important role in their
presence or absence. In a study utilizing granular activated carbon (GAC), it was determined that the
autochthonous microbial community influenced pathogen survival (2). When pathogens were introduced to
sterile GAC in the presence of heterotrophs, they attached at levels similar to those found in pure culture,
then decreased. However, when the two were added to GAC with a mature biofilm, the pathogens attached
at lower levels and decreased at a more rapid rate.
Future research will address the enumeration of bacterial pathogens as it relates to bioftlm development
on activated carbon, similarly, efforts will continue in the characterization of fecal streptococci and enteric
viruses associated with the wastewater treatment system.
ACKNOWLEDGMENT
This work was supported by funding from the Technology Utilization program, National Aeronautics and
Space Administration.
109
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4.
LITERATURE CITED
Antonie, R. L.: Fixed Biological Surfaces - Wastewater Treatment. CRC Press, Inc., (West Palm
Beach), 1978.
Camper, A. K.; LeChevallier, M.W.; Broadaway, S. C., and McFeters, G.A.: Growth and Persistence
of Pathogens on Granular Activated Carbon Filters. Appl. Environ. Microbiol. vol 50, 1985, pp.
1378-1382.
Cox, C. S.: The Aerobiological Pathway for Microorganisms. John Wiley and Sons (New York), 1987.
Johnson, A. H.; Bounds, B. K.; and Gardner, W.: I. Assessment of Internal Contamination Problems
Associatedwit_Bioregenerative Air/Water Purification Systems. SAE Technical Paper Series,
Proceedings of the 20th Intersociety Conference on Environmental Systems, Williamsburg, VA., July
9-12, 1990.
5. A.P.H.A. Standard Methods for the Examination of Water and Wastewater, 17th Edition, 1989.
6. Block, J. C.; and Schwartzbrod, L.: Detection and identification of Viruses in Water Systems. VCH
Publishers (New York), 1989.
7. Brock, T. D.; Smith, D. W.; and Madigan, M. T. Biology of Microorganisms. 4th Edition.
Prentice-Hall, Inc., (Englewood Cliffs, NJ), 1984.
8. Gersberg, R. M.; Gearheart, R. A.; and Ives, M.: Pathogen Removal in Constructed Wetlands. In D.
A. Hammer (ed.), Constructed Wetlands for Wastewater Treatment: Municipal, Industrial and
Agricultural, pp. 431-445. Lewis Publishers, Inc., (Chelsea, MI), 1989.
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Gersberg, R. M., Lyon, S. R.; Brenner, R.; and Elkins, B. V.: Fate of Viruses in Artificial
Wetlands. Appl. Environ. Microbiol. vol 53, 1987, pp. 731-736.
:=
Hammer, D. A.; and Bastian, R. K.: Wetland Ecosystems: Natural Water Purifiers? I..._nD. A. Hammer
(ed.), Constructed Wetlands for Wastewater Treatment: Municipal, Industrial and Agricultural, pp.
2-19. Lewis Publishers, Inc., (Chelsea, MI), 1989.
Kadlec, J. A.: Nutrient Dynamics in Wetlands. I...qnK. R. Reddy and W. H. Smith (ed.), Aquatic Plants
for Water Treatment and Resource Recovery, pp. 393-419. Magnolia Publishing, Inc., (Orlando),
1987.
Armstrong, W.: Oxygen Diffusion From the Roots of British Bog Plants. Nature vol 204, 1964, pp.
801-802. :_: ........ :...........
Good, B. J.; and Patrick, W. H.: Root-Water-Sediment Interface Processes. I..._nK. R. Reddy and W.
H. Smith (ed.), Aquatic Plants for Water Treatment and Resource Recovery, pp. 359-372. Magnolia
Publishing, Inc., (Orlando), 1987.
B0wen, G. D. ; and Rovira, A. D. : Microbial Colonization of Plant Roots. Annu.
vol 12, 1976, pp. 181-197.
Rev. Phytopath,
Broadbent, P.; Baker, K. F.; and Waterworth, Y.: Bacteria and Actinomycetes Antagonistic to Fungal
Root Pathogen in Australian Soils. Aust. J. Biol. Sci. vol 24, 1971, pp. 925-944.
Palmateer, G. A.; Kutas, W. L.; Walsh, M. J., and Koellner, J. E.: Abstracts of the 85th Annual
Meeting of the Am. Soc. for Microbiol. Las Vegas, NV, March 3-7, 1985.
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Seidel, K.: Macrophytes and Water Purification. In J. Tourbier and R. W. Pierson, Jr. (ed.),
Biological Control of Water Pollution, pp. 109-121. University of Pennsylvania Press (Philadelphia),
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Gersberg, R. M.; Brenner, S. R.; Lyon, S. R.; and Elkins, B. V.: Survival of Bacteria and Viruses in
Municipal Wastewater Applied to Artificial Wetlands. InnK. R. Reddy and W. H. Smith (ed.),
Aquatic Plants for Water Treatment and Resource Recovery, pp. 237-246. Magnolia Publishing, inc.,
(Orlando), 1987.
111
TABLE 1
BIOHOME MEAN MONTHLY BOD VALUES
DATE INFLUENT (mg/L) EFFLUENT (mg/L}
6/89 368 1.2
7/89 264 2.0
8/89 217 20.6
9/89 388 3.8
10/89 293 7.2
11/89 304 2.0
12/89 245 2.2
1/90 114 1.8
2/90 234 7.2
3/90 236 1.5
4/90 224 11.6
5/90 357 3.1
TABLE 2
BIOHOME MEAN MONTHLY FECAL COLIFORM COUNTS
DATE INFLUENT EFFLUENT
6/89 4.8 X 105 1
7/89 1.5 X 105 1
8/89 8.0 X 106 800
9/89 4.4 X i06 6000
10/89 8.5 X 105 8000
11/89 4.0 X 104 1
12/89 8.0 X 105 6000
1/90 4.2 X 104 530
2/90 8.0 X 104 10
3/90 8.0 X 104 1
4/90 2.5 X 104 150
5/90 8.0 X 105 1
TABLE 3
BIOHOME FECAL STREPTOCOCCI DENSITIES (CFUs/100 ml)
WEEK # INFLUENT EFFLUENT
1 58 0
2 57 0
3 59 0
4 45 0
5 56 0
6 50 0
7 57 0
8 49 0
9 50 0
i0 52 0
112
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ENVIRONMENTAL AND FACILITIES MANAGEMENT SYSTEM
Bruce Davis
Geographer _
Stennis Space Center
SSC, MS 39529
__ 5 _ __ -- _
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