Biological Treatment of Space Habitation Waste Waters

49th International Conference on Environmental Systems ICES-2019-341 7-11 July 2019, Boston, Massachusetts

Copyright © 2019 Pancopia, Inc.

Biological Treatment of Space Habitation Waste Waters

using a Two Stage Reactor

Bill Cumbie1, John Whitelaw 2, Fei Dai3, Holly Anne Matel

Pancopia, Inc., Hampton, VA, 23666

W. Andrew Jackson4

Texas Tech University, Lubbock, TX, 79407

The development of full-size membrane-aerated bioreactors (MABRs) (CoMANDR 1.0,

CoMANDR 2.0, and rCoMANDR) to achieve aerobic biological stabilization has been

previously demonstrated over operating periods of ~1 year. These systems have successfully

treated a variety of possible habitation waste streams including compositions likely to be

encountered in space-based exploration including both space station and planet-based flows.

The effluent of the MABR has good stability and is low in organic carbon. The MABR

removes approximately half of the ammonia nitrogen in the wastewater and converts half of

the remaining ammonia to nitrite, making it an attractive candidate for a second stage anoxic

reactor using anammox bacteria. To test this hypothesis, six anoxic anammox bioreactors

were operated for a three-month period to determine if they could successfully remove

ammonia and nitrite from MABR effluent wastewater, generated from the EPB waste stream.

The reactors achieved 85% nitrogen removal and operated stably over the test period.

Nomenclature

Annamox = Anaerobic Ammonium Oxidizing bacteria

COD = Chemical Oxygen Demand

DO = Dissolved Oxygen

DOC = Dissolved Organic Carbon

EPB = Early Planetary Base

ISS = International Space Station

MABR = Membrane Aerated Biological Reactor

NASA = National Aeronautics and Space Administration

NOx = Oxidized nitrogen species, nitrite and nitrate

ORP = Oxidation-Reduction Potential

PCS = Process Control System

rCOMANDR = rectangular COunter-diffusion Membrane Aerated Nitrifying Denitrifying Reactor

TN = Total Nitrogen

TOC = Total Organic Carbon

TTU = Texas Tech University

USDA = United States Department of Agriculture

I. Introduction

Maintaining the conditions for healthy human habitation is a key component of space exploration, both in transit

and in eventual extra-terrestrial settlements. Of the inputs for life-support, water represents a minimum of two-thirds

the daily mass needed to sustain astronauts in space. Although short missions can be provisioned entirely by initial

1 Chief Executive Officer, Pancopia Inc. 1100 Exploration Way, Hampton, VA 23666 2 Chief Technical Officer, Pancopia Inc. 1100 Exploration Way, Hampton, VA 23666 3 Lab Manager, Pancopia Inc. 1100 Exploration Way, Hampton, VA 23666 4 Professor and Associate Chair, Civil and Environmental Engineering, MS 41023

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International Conference on Environmental Systems

water reserves, longer missions require in-situ recycling of water. Current systems for water recovery at the

International Space Station (ISS), are based on physical-chemical methods, and require significant input of energy

and consumables, including hazardous chemicals used to stabilize urine.1 These systems achieve approximately 70%

water recovery2. As missions become longer and more remote from earth, increased water recovery rates and reduced

consumable inputs will be indispensable due to the limitations on resupply. NASA continues to invest in the

development of new, lower-cost solutions to recycle and reuse water for future manned space exploration efforts.

Several biological systems have been investigated for treatment of space wastewater, with considerable success.

Texas Tech University (TTU) has experimented with variations on a membrane-aerated bioreactor since 2002. Its

most recent iteration, their rectangular Counter-diffusion Membrane-Aerated Nitrifying-Denitrifying Reactor

(rCOMANDR) unit, is an aerobic process which treats space wastewater at a rate equivalent to that produced by 2

crew per day, achieving >50% conversion of organic nitrogen to ~90% NO2- and 10% NO3

-, and >85% removal of

dissolved organic carbon (DOC)3. The effluent ratio of NH4+: NO2

- is typically near 1:1, making it potentially well

suited to further processing by anaerobic ammonium oxidizing (anammox) bacteria, which use ammonium and nitrite

in a stoichiometric ratio of approximately 1:1.3.

Predicted in 1977 by Broda4 and first isolated in 19955, anammox bacteria has revolutionized nitrogen removal

from wastewater. It has been identified as the major new wastewater treatment technology of the century6 and can

remove nitrogen at one-third the cost of

traditional nitrification-denitrification

technology. Anammox organisms use nitrite to

oxidize ammonia in anoxic conditions, with

nitrogen gas as the primary product.

These attributes make it an ideal candidate

to polish the effluent from a membrane-aerated

bioreactor (MABR) that produces a fifty-fifty

mix of ammonium and nitrite, such as the Texas

Tech rCOMANDR unit. Pancopia, Inc. teamed

with TTU to determine if a two-stage biological

reactor could produce a superior effluent for

recycling of water for space missions.

II. Materials and Methods

Since 2015, researchers at TTU have been

evaluating the ability of a rectangular MABR,

rCOMANDR, to treat habitation waste streams.

rCOMANDR is designed in a rectangular

configuration to treat the wastewater produced

by 2 crew per day (Figure 1). The system is

micro-gravity compatible, as it does not

produce 2-phase flow (air/water) but instead

relies on diffusion of O2 through siloxane tubes

on which biofilm grows. The system is designed

to be able to convert dissolved organic carbon

(DOC) to dissolved CO2 or cell mass, organic N

to nitrite and nitrate (NOx-), and to lower the pH

when operated in an aerobic mode (dissolved

oxygen (DO) >5 mg/L)3.

Texas Tech researchers have evaluated

reactor operation for various waste streams (Early Planetary Base (EPB), Transit, and ISS, see Table 1) at a constant

loading rate equivalent to 2 crew per day. For all test points, reactor influent and effluent were evaluated daily for total

DOC, total nitrogen (TN), NO2-, and NO3

-.

To date the system has been in operation for > 2.5 years and has treated a total of 12,000 L. During this time, no

biosolids have been removed. The system is able to achieve >85% DOC removal and >50% conversion of organic N

to NOx- with little TN removal (~10%) (Table 1). For most test points NO2

- is the dominant form of NOx-, although

rCOMANDR also produces small amounts of NO3-. Effluent pH ranges from 5 to 7 depending on the influent waste

stream3.

(b)

(a)

Figure 1. Schematic design (a) and photo during

operation (b) of rCOMANDR unit.

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Table 1. Results of rCOMANDR Operation for Various Waste Streams.

Waste

Stream

DOC (mg/l) TN (mg/l) NOx- (mg/l) NO2

- (%) pH

Influent Effluent Influent Effluent Effluent NO2/NOx

EPB 770 110 900 740 380 91 6.8

Transit 990 130 700 750 400 88 5.6

ISS 2500 160 2900 2400 1200 85 6.2

(a)

(b)

Figure 2. Phase I Reactor design. Section view (a) and plan view (b).

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A. Second-Stage Reactor Design

The reactor design was based on reactors used previously by Pancopia, Inc. with mixed-organism treatment

(nitrifiers, denitrifiers, and anammox) to remove organic carbon and nitrogen7. This design was effective in treating

ersatz EPB wastewater, demonstrating that the arrangement of media, organisms, and circulation/heating equipment

in the system can provide effective process control, adequate substrate transport, and a hospitable environment to the

organisms. The foam media is critical to the effective retention of slow-growing anammox biomass in a continuous-

flow treatment system since the organisms colonize the media and are protected within from shear forces associated

with mixing and from washout by feed/withdrawal flows. The design of the foam media blocks in the intermittently

aerated, mixed-organism reactors in previous research7 used higher-density foam throughout (30 pores per inch (ppi)

foam center and 20 ppi foam exterior) to promote the creation of an anoxic zone inside the center region of the media.

For the purely anaerobic anammox reactor, lower density foam (10 ppi) was used throughout the media, to allow both

faster substrate transport and more extensive microorganism deposition into the full depth of the foam.

Each reactor consisted of a 20 L-capacity rectangular vessel of approximately 48 cm x 15 cm x 25 cm, operated at

an effective volume of 15 L. A Styrofoam cover was constructed to float on top of the water surface, minimizing

oxygen transfer from the atmosphere and evaporation of liquid from the reactor. Penetrations were cut in the cover to

allow insertion of probes, influent and effluent hoses, nitrogen gas lines, equipment power cables, and for removal of

liquid samples. Two, 13 cm x 13 cm x 8 cm, foam scaffolds were arranged in a line in the center of the reactor, with

mixing pumps circulating the bulk fluid around them. A heater maintained temperature at 34°C during operations,

based on the operating set point of the United States Department of Agriculture (USDA) batch testing experiment8.

N2 gas diffusers were used during initial setup to sparge oxygen from the reactor. Sparging was repeated during

operations as needed to maintain a DO of less than 0.2 mg/L, usually only when covers were removed for maintenance

activities and oxygen was introduced.

Process control and monitoring was managed with Neptune Apex process control systems (PCS). These included

sensors to monitor pH, oxygen-reduction potential (ORP), conductivity, and temperature. Additionally, three Water

Analytics AM-ODO optical DO probes were connected to the Apex PCS in reactors 4 to 6. In reactors 1 to 3, YSI

FDO® 70x IQ optical DO probes were used and connected to a YSI Sensornet 2020xt PCS for monitoring. DO, pH,

ORP, conductivity, and temperature were continuously monitored during operation and testing. The Neptune Apex

PCS also allowed both local and remote manual and automated control of pumps, N2 gas supply, and heaters, which

promoted stability and reliability in operations.

Testing of the mechanical and electronic components prior to addition of organisms was performed. The system

was tested prior to inoculation with anammox, to ensure that dissolved oxygen could be maintained below 0.2 mg/L

while mixing and feeding/withdrawing fluid.

Figure 3. Phase I reactor in operation.

Figure 4. Setup of six second-stage anammox reactors.

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B. Anammox culture to seed reactors

Pancopia, Inc. cultured the anammox biomass for inoculation in the reactors in a 40L continuously mixed anoxic

bioreactor, pictured in Figure 5. Seed culture of Candidatus Brocadia Caroliniensis (Accession Deposit Number

NRRL B-502869) was provided by USDA Agricultural Research Service in Florence, SC, where this strain was

isolated.

The six anammox bioreactors were inoculated directly from Pancopia’s culturing reactor initially with 12.5 g total

suspended solids (TSS) of anammox each, then an additional 5.7 g TSS was added to each reactor during week four

of operation.

C. rCOMANDR effluent from Texas Tech University

Texas Tech University provided 440 L of effluent from their rCOMANDR bioreactor, operated in fully-aerobic

conditions. Effluent was generated and shipped on a bi-weekly schedule in sealed coolers to minimize any degradation

prior to treatment in the test reactors. When a batch of effluent was in use as feed, it was sampled at least weekly to

monitor changes in its characteristics. Effluent batches not in use as feed were stored in a freezer to prevent

degradation.

III. Results

Six second-stage anammox reactors were operated for twelve weeks to determine the feasibility of using this

process for enhanced recycling of wastewater generated during space missions. Feasibility was defined as the

achievement of an average removal rate of nitrite and ammonium >50% over a four-week test period, in two out of

three test reactors. The test of feasibility was conducted during the final four weeks of the operational period. From

the six reactors, the three best-performing were selected as test reactors. These were operated at pH 7.3, temperature

34oC, with continuous mixing.

All three test reactors exceeded the feasibility criteria of over 50% removal of both ammonium and nitrite, with

average removal for the three reactors during the test period of 64% ammonium and 78% nitrite. The three lower-

performing reactors also met the >50% removal target for both constituents during the test period, indicating that this

process can be an effective second-stage treatment for rCOMANDR effluent, significantly reducing residual nitrogen

in the waste stream.

In the last four weeks of the operational period, the nitrite and ammonium removal rates in the reactors began to

decline. The cause of this instability remains unclear, but it may have been a result of partial inhibition of the anammox

organisms by elevated nitrite exposure, or due to a growing culture of denitrifying organisms competing with the

anammox for nitrite substrate.

Figure 5. Pancopia's 40L anammox

culturing reactor.

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A. Ammonium and nitrite removal

The second-stage reactors demonstrated excellent ammonium and nitrite removal rates in the first 8 weeks of the

operational period, with removal efficiencies of nitrite between 90% and 100% and of ammonium between 75% and

95%. Differences in the removal rates of the two substrates are a consequence of the reaction stoichiometry: In a pure

anammox process, ammonium and nitrite removal rates are related by the stoichiometry of the reaction, approximately

1 NH4+:1.3 NO2

-. If the ratio of these two substrates in the feed is not matched to the reaction, one or the other substrate

can be rate-limiting. In the case of the rCOMANDR effluent, NH4+:NO2

- varied considerably over the course of this

study, as is shown in Figure 7. Typically, ammonium concentrations in the influent to the second-stage reactors were

equal to or higher than nitrite concentrations, resulting in a nitrite-limited anammox process. Ammonium

concentrations in the second-stage effluent were higher than nitrite throughout the operational period as a result. This

effect is particularly clear in the period following 11-6-18, when ammonium in the influent increased significantly

relative to nitrite. The effect is also visible in the removal efficiencies calculated for nitrite and ammonium in the

three test reactors over the course of the study (Figure 6). Nitrite removal rates are higher throughout the study period.

Figure 7 Influent and average effluent concentrations of ammonium

and nitrite over study period. (Note that influent to second-stage reactor

is rCOMANDR effluent.)

(b) (a)

Figure 6. Ammonium (a) and nitrite (b) removal efficiencies, three test reactors.

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B. Decline in Mass Removal Rate

The last four weeks of the operational period, coincident with the feasibility test period, showed a consistent

decline in the removal efficiencies of ammonium and nitrite (see Figure 6). Examination of the mass removal rates

of both substrates (Figure 8) shows that there was a decline of approximately 30% and 48% in average rates of NH4+

and NO2- removal, respectively, between the first 8 weeks and the last 4 weeks of the study. With a reduced mass

conversion rate and a relatively constant loading rate, the constituents accumulated in the reactors and the overall

removal efficiency declined rapidly.

Although the exact cause of the decline in performance in the last 4 weeks of operations is not clear, two primary

hypotheses have been identified: 1) partial nitrite inhibition led to reduced anammox activity, and 2) low levels of

organic carbon in the feed were sufficient to promote the growth of a culture of denitrifiers which competed with

anammox for nitrite.

1. Nitrite inhibition

Although nitrite is an essential substrate for the anammox metabolism, multiple studies have demonstrated that

elevated levels of nitrite can cause inhibition of anammox activity10. Reported inhibition thresholds vary between 5

and 280 mg/L NO2-N, depending on the strain of anammox organism involved and the configuration of the system.

The anammox strain used in this study, C. Brocadia Caroliniensis, has been cultured successfully at nitrite levels of

140 mg/L9. However, in other work at Pancopia’s facilities, it has been found to perform best with concentrations of

nitrite below 30 mg/L NO2-N. Furthermore, researchers have found that nitrite inhibition threshold can increase with

time as anammox becomes acclimated.11 Based on these factors, nitrite levels were closely monitored during the first

eight weeks of continuous operation and the reactors were diluted with deionized water when NO2-N accumulated to

between 30 and 50 mg/L. Dilution was not used during the feasibility test period to avoid distorting the removal

efficiency data, and accumulation of nitrite during this period may have caused some inhibition. A positive feedback

loop would then have begun, where higher nitrite caused a reduced metabolic rate, leading to even higher nitrite

concentrations.

Figure 8. Mass removal rates and reactor concentrations of ammonium and

nitrite through the operating period. Average removal rates are calculated for the

periods before and after the start of the test period.

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Ammonium removal rates were plotted against nitrite

concentrations for the whole operating period to evaluate

the possibility of nitrite inhibition as a factor in reactor

performance degradation (Figure 9). There is a weak

correlation between lower concentrations and higher

removal rates, and the removal rate appears to decline at

NO2-N above 20 mg/L. The highest nitrite concentrations

were observed at the end of the study period, and may

reflect acclimation occurring, since the metabolic rate at

these concentrations (2.9 to 4.9 mg/h) appears to be

recovering to nearer the rates observed at <20 mg/L NO2-

N.

2. Denitrifier competition

Examination of the measured ratios of ammonium and

nitrite consumption as well as nitrate production in the

reactors shows a change between the stable performance

period prior to the feasibility test and the declining

performance in the last four weeks. The stoichiometry of

the anammox reaction9 is

1 𝑁𝐻4+ + 1.3𝑁𝑂2

− → 0.18𝑁𝑂3− + 1.06𝑁2 (1)

In the period of stable operations,

stoichiometry was close to the theoretical

ratio, with 1 NH4+:1.2 NO2

- consumed: 0.03

NO3- produced (see Figure 10). However, as

the performance started to decline,

ammonium was consumed at a higher rate

than nitrite in the overall reactor system (1

NH4+:0.8 NO2

-: 0.07 NO3- consumed). Since

there is no known mechanism for anaerobic

ammonium oxidation besides the anammox

process, and the dissolved oxygen in the

reactors was always <0.1 mg/L, some other

anaerobic reaction must have been generating

nitrite in the system to replace the nitrite

consumed in ammonium oxidation.

Biologically mediated partial denitrification

from nitrate to nitrite is a likely candidate for

this reaction. Corroborating this hypothesis,

nitrate in the final four weeks of operations

showed net consumption rather than

production. Biological denitrification

reduces nitrite and nitrate using organic

carbon as the terminal electron acceptor,

therefore organic carbon should also be

consumed in the reactors if partial denitrification was occurring. Although COD was removed in the reactors during

the entire operational period, the mass ratio of COD to NO2-N removal increased from 0.77 during stable operations

to 1.2 in the last four weeks, indicating an increased rate of denitrification.

The second-stage reactors were designed as a pure anammox system, using environmental conditions to promote

this organism over other competitors. In this case, it was expected that anoxic conditions, plentiful ammonium and

nitrite substrate, and an influent stream almost completely free of available organic carbon would ensure that the

anammox culture dominated; however, it appears that carbon in the rCOMANDR effluent was more biologically

available than anticipated, and a denitrifying population developed. It is likely that with increasing denitrifier biomass,

anammox was in competition for available nitrite, resulting in lower overall anammox activity.

Figure 10. Ratios of average NH4 and NO2 removal rates, NO3

production rates during periods of stable and declining

performance, compared to theoretical anammox stoichiometry.

Negative values indicate production.

Figure 9. Nitrite concentration vs. ammonia

removal rate. Data from entire operational period.

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IV. Possible Future Research

Pancopia proposes to continue development of a two-stage biological wastewater processor in partnership with

Texas Tech in 2019. This work would begin with a more detailed investigation of sources of instability in the

anammox process as applied to rCOMANDR effluent. Once boundary conditions for nitrite concentration and

available organic carbon in the influent are identified, a scaled-up anammox reactor will be developed integrating a

preconditioning module designed to adjust nitrite/ammonium ratios and to remove excess organic carbon. The scaled-

up reactor will be tested on its own to identify the stable operating space for the process in terms of temperature, pH,

nitrite and ammonia concentrations. Furthermore, the second-stage reactor will be integrated with an rCOMANDR

unit and the overall system stability as well as its response to variations in flow characteristics and loading will be

investigated.

V. Conclusion

Operation of second-stage anammox reactors to treat effluent from TTU’s rCOMANDR unit has demonstrated

that the anammox process can be effective in removing a significant portion of remaining nitrogen in this waste stream.

Over 12 weeks, the reactors averaged between 79% and 86% removal of ammonium and nitrite, achieving 80% total

inorganic nitrogen removal overall. The process encountered some instability in the last four weeks of operation,

likely due to either partial nitrite inhibition or competition from denitrifying bacteria or both. Further work is needed

to investigate in detail the causes of instability and identify the operating space (pH, temperature, NH4+ and NO2

-

concentrations) within which the process will treat reliably. Design of a larger-scale anammox reactor with higher

treatment density will be needed to integrate with the rCOMANDR module in a two-stage system. Additionally, the

overall two-stage system response to variation in waste stream quantity and quality should be investigated.

Acknowledgments

The authors wish to acknowledge Dr Matias Vanotti of USDA-ARS and Dr Kevin Gilmore of Bucknell University

for their expert advice and consultation on this project as well as the technicians at USDA-ARS for their chemical

analysis support provided through a Cooperative Research and Development Agreement between Pancopia Inc. and

USDA who also licensed the patented strain of anammox used in this study. Dr. Andrew Jackson and Texas Tech

University provided the effluent from the first stage biological reactor and provided senior consultation. The authors

acknowledge the funding that made this work possible. This work was supported by NASA Small Business Innovation

Research contract 80NSSC18P1954.

References

1. Sevanthi, R., D. Christenson, E. Cummings, K. Nguyen, A. Morse, et al. Performance of a Full Scale MABR (CoMANDR

2.0) for Pre-treatment of a Habitation Waste Stream Prior to Desalination. 2014. 44th International Conference on Environmental

Systems.

2. Christenson, D., R. Sevanthi, A.N. Morse, and W.A. Jackson, Assessment of Membrane-Aerated Biological Reactors

(MABRs) for Integration into Space-based Water Recycling System Architectures. Gravitational and Space Research, 2018. 6(2).

3. Jackson, W.A., Summary memo on rCOMANDR operation and performance, I. Pancopia, Editor. 2018. p. 2.

4. Broda, E., Two kinds of lithotrophs missing in nature. Zeitschrift für allgemeine Mikrobiologie, 1977. 17(6): p. 491-493.

5. van de Graaf, A.A., A. Mulder, P. de Bruijn, M.S. Jetten, L.A. Robertson, et al., Anaerobic oxidation of ammonium is a

biologically mediated process. Applied and Environmental Microbiology, 1995. 61(4): p. 1246.

6. Murthy, S. Unlocking the mysteries of mainstream deammonification - A paradigm shift in the wastewater industry.

Excellence in Environmental Engineering and Science™ Awards Competition Winner 2013 [cited 2019 April 29, 2019]; Available

from: http://www.aaees.org/e3scompetition/2013grandprize-research.php.

7. Pancopia, NASA Phase II SBIR Proposal: Rapid Activation of Biological Wastewater Treatment Systems. Prop. No. 15-

1 H3.02-911. 2015.

8. Vanotti, M., Preliminary batch testing of anammox bacteria effectiveness in treating rCOMANDR effluent (unpublished

report). 2018.

9. Vanotti, M.B., A.A. Szogi, and M.J. Rothrock, Anammox bacterium isolate. 2013, Google Patents.

10. Jin, R.-C., G.-F. Yang, J.-J. Yu, and P. Zheng, The inhibition of the Anammox process: A review. Chemical Engineering

Journal, 2012. 197: p. 67-79.

11. Kimura, Y., K. Isaka, F. Kazama, T.J.A.m. Sumino, and biotechnology, Effects of nitrite inhibition on anaerobic

ammonium oxidation. 2010. 86(1): p. 359-365.

http://www.aaees.org/e3scompetition/2013grandprize-research.php

Documents

Biological Treatment of Space Habitation Waste Waters