SEPA
                            United States
                            Environmental Protection
                            Agency
EPA/540/S-93/505
October 1993
                           SUPERFUND INNOVATIVE
                           TECHNOLOGY  EVALUATION
                             Emerging  Technology
                            Summary
                             Pilot-Scale   Demonstration  of  a
                            Two-Stage   Methanotrophic
                             Bioreactor  for  Biodegradation   of
                            Trichloroethene in  Groundwater
                              BioTrol, Inc., developed a two-stage,
                            methanotrophic, bioreactor system for
                            remediation of water contaminated with
                            trichloroethylene (TCE) and other chlo-
                            rinated, volatile, aliphatic hydrocarbons.
                            The first stage was a suspended-growth
                            culture vessel with a bubbleless meth-
                            ane transfer device. The second stage
                            was a plug-flow reactor fed with con-
                            taminated groundwater and effluent
                            from the culture vessel. The system
                            was tested at bench- and pilot-scale.
                            When operating optimally, 89% of the
                            influent TCE was degraded. Reactor ki-
                            netics were consistent with first-order
                            biodegradation kinetics. Actual meth-
                            ane use in  the pilot-scale reactor re-
                            sulted in projected methane costs of
                            $0.33 per 1000 gal of water treated.
                            This cost could be reduced by modifi-
                            cations to the system. Calculated theo-
                            retical minimum methane costs were <
                            $0.05 per 1000 gal.  Variability in the
                            degree of TCE degradation and diffi-
                            culty in maintaining the activity of the
                            microbial culture during  continuous
                            operation were noted. Sustained use of
                            the technology will  require modifica-
                            tions to culture conditions.
                              This Summary was developed by
                            EPA's Risk Reduction Engineering
                            Laboratory, Cincinnati, OH,  to announce
key findings of the SITE Emerging Tech-
nology program that is  fully docu-
mented in a separate report (see Project
Report ordering information at back).

Introduction
  Chlorinated, volatile, aliphatic hydrocar-
bons (Clx-VOCs) are the most commonly
reported contaminants  of groundwater. The
reason for their widespread occurrence in
the environment is their widespread use
as solvents and degreasers. Since this
problem came to light as recently as the
early 1980s few approaches have been
developed for remediating TCE-contami-
nated sites. Currently available remediation
methods for subsurface environments in-
clude air sparging of the  groundwater,
vacuum extraction of contaminants from
the vadose zone, and extraction of con-
taminated water for  air-stripping. These
techniques transfer contamination from the
subsurface environment to either the air
or to activated carbon, which must then
be landfilled or incinerated. Landfilling the
contaminated activated carbon  transfers
the contamination to another environment,
and incineration is costly and requires con-
siderable energy and capital equipment to
completely  oxidize  volatile chemicals.
Treatment systems based on oxidation of
contaminants that use ultraviolet radiation
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in combination with  a  chemical oxidant
(peroxide  or ozone)  are  also available,
but these methods are energy  intensive
and  require addition of  expensive chemi-
cals.
  A  number of particularly promising new
approaches rely on  bacterial  cooxidation
of the Clx-VOCs during growth on another
(primary) carbon source. One such bacte-
rium is the   obligate  methanotroph
Methylosinus trichosporium OB3b  (here-
after, M.t. OB3b). This organism  produces
soluble  methane monooxygenase (sMMO)
when grown on single-carbon substrates
(methane,  methanol,  or formate). SMMO
is an enzyme of  low substrate specificity
capable of  catalyzing a variety  of oxida-
tion  reactions in addition to the  oxidation
of methane. Among those  reactions  are
the oxidations  of several  Cl -VOCs  (see
Table 1 for  list)-reactions often resulting
in stoichiometric quantities of mineral end
products (carbon dioxide, water,  and chlo-
ride  ion).
  The full   report  addresses  the  use of
M.t. OB3b  for  remediation of TCE-con-
taminated groundwater with the  use of a
two-stage bioreactor.  In this system,  cells
produced at high  concentration  in a cul-
ture   medium  contacted  contaminated
groundwater in a  plug-flow reactor. The
objectives of the study were:
  . to determine reactor  design parameters
     at bench scale and  to operate a  pilot-
     scale reactor  to achieve degradation
     of TCE and
  . to  determine operating  values  for
     parameters  that  influence the
     economic  competitiveness  of  the
     system.
   Since the economic viability of the sys-
tem  was dependent  on the efficiency of
     methane utilization  in the culture vessel,
     an  innovative methane  transfer  method
     was used  to  increase  methane  transfer
     efficiency.

     Procedure
       In bench-scale  experiments, the  con-
     ceptual design was  evaluated and starting
     values for  the operational  parameters of
     the bioreactor were determined. After the
     concept was confirmed  at the bench,  a
     pilot-scale  reactor  that  used  the  design
     criteria established  during the  bench  tests
     was constructed.
       For the bench-scale system, cells  were
     grown in a 2000 ml chemostat vessel in
     1000 ml of culture medium  and fed to
     fabricated glass columns where they con-
     tacted contaminated water. The total flow
     rate was adjusted by adding make-up wa-
     ter. Influent and effluent TCE concentra-
     tions were measured over  the  plug-flow
     reactor  before and after initiation of cell
     culture flow to the column. Thus,  conser-
     vation of TCE was established before in-
     troducing the cells.
       For the  pilot test, groundwater treated
     by  air-stripping to  remove TCE was ob-
     tained from a nearby army munitions  facil-
     ity  and carried  in  a stainless-steel  tank
     truck to BioTrol's pilot testing  facility. The
     water was  then piped from the truck to a
     500 gal,  polyethylene surge  tank and me-
     tered into a stainless-steel plug-flow  reac-
     tor  at a controlled  rate. A high-concentra-
     tion TCE solution  (prepared in degassed
     distilled water) was metered into the  influ-
     ent groundwater upstream from the  influ-
     ent sampling port. The TCE solution was
     held in a  Teflon gas-sampling  bag that
     collapsed as the TCE  solution was pumped
     out. TCE and cells  were added within the
 Table \ Summary of Compounds Degraded by Methylosinus trichosporium  0636
 Methanes
 dichloro (methylene chloride}
 trichloro (chloroform)
 Ethanes

 1,1-dichloro
 1,2-dichloro
 1,1,1-trichloro
Ethenas

chloro (vinyl chloride)
1,1-dichloro (vinylidene chloride)
t-1,2-dichloro (DCE)
c-1.2-dichlom(DCE)
trichloro (TCE)

Other

1,3-dichioropropene (-propylene)
2,2,2-tr/chloroacetaldehyde (chloral hydrate)
closed reactor system to avoid TCE losses
by volatilization. Once again, TCE conser-
vation was established before bacteria
were  introduced to the plug-flow reactor.
  Culture medium containing a high den-
sity of cells (Asoo = 1.8,  or approximately
32 mg dry ceils/L) was pumped into the
plug-flow reactor at a  rate equivalent to 1/
10 the rate of groundwater flow. The total
flow to the plug-flow reactor was 1 L/min.
(See  Table 2 for operating  parameters.)
The  TCE  concentration  was  measured
over the full length of the plug-flow reactor
(at approximately 20-ft intervals).  Reactor
performance was determined  on the  basis
of TCE concentrations throughout the re-
actor. Growth and activity of  the microor-
ganisms  within the  culture vessel   were
evaluated on the basis of culture density,
color  (visually),  and  sMMO  activity with
the use of a colorimetric assay. All  materi-
als contacting the contaminated water were
either stainless steel,  Teflon,  or glass.

Results and Discussion
  A flow diagram of the reactor system is
provided in  Figure 1,  and the operational
parameters  for the bench tests  are shown
in Table 2. The intention of the  bench test
was to  determine parameters that  would
provide for  stable, continuous treatment
of TCE by M.t.  OB3b.   A culture-vessel
dilution  rate  of 0.02/hr  was  established
experimentally to maintain a  steady-state
M.t.  OB3b  concentration  based on the
growth  rate  of  the  bacteria in  the
chemostat.  Growth of the organisms is,
however,  a  function of methane availabil-
ity,  which  is,  in  turn, a function of gas
transfer efficiency. Gas  transfer efficiency
is variable based on the chemostat's char-
acteristics  (aerator and impeller  dimen-
sions, etc.)  and,  thus, will change during
scale-up.
  TCE biodegradation over several hours
of treatment with the  use of the optimized
bench-scale  reactor  system  is illustrated
in Figure  2. Average  influent and effluent
TCE concentrations were  563  and 63 parts
per billion  (ppb), respectively,  which cor-
respond to an 89% TCE reduction.
  Although  these results signify an out-
standing  potential  for  TCE  treatment
through the reactor, instability of the pure
culture of A/I. f. OB3b was noted. This sug-
gests  that  ultimate  modifications  to the
culture system would  be  needed to achieve
stable, long-term  treatment. The instability
was noted as a sharp decrease in sMMO
activity by  colorimetric assay followed by
change in the color  of  the  culture from
yellow to dull green color. A mixed culture

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that included  M.t. OB3b,   various  other
morphologically diverse bacteria,  and an
abundance of ciliates 'was observed in the
culture medium by phase-contrast micros-
copy. To achieve continuous treatment of
TCE, this  problem must  be  addressed;
however,  to  determine the feasibility of
the treatment concept before added effort
was given to culture development, the pi-
lot demonstration proceeded.
  The primary objectives of the pilot-scale
demonstration  were  (1)  to determine
whether TCE  degradation  activity similar
to that observed during the  bench test
could be induced at pilot scale and  (2) to
evaluate the  costs of operating the reac-
tor system to treat TCE-contaminated wa-
ter. Because the cost of methane gas was
a primary concern, a bubbleless, gas trans-
fer device was  added to the reactor sys-
tem to increase the methane transfer effi-
ciency (Figure  1).  Culture  medium was
circulated through the device for methane
saturation and then returned to the culture
vessel.
  When  operating optimally, 88% of the
influent TCE was biodegraded by  using
the operating parameters shown in  Table
2. TCE concentrations were measured at
various distances down the plug-flow re-
actor to  compare actual data to a first-
order kinetic model. The results are shown
in Figure  3.  Clearly, with optimal perfor-
mance, the first-order model  adequately
describes TCE removal from the contami-
nated water.
  TCE disappearance  was  monitored
through the  reactor  system  during two
separate operations. Operations 1 and  2
lasted  10  and 8  days, respectively. Some
degree of TCE treatment (minimum 14%)
was accomplished on each day  of each
operation. Typically, 5 or 6 days  of near-
optimal reactor performance (and  approxi-
mately first-order  reactor  kinetics) were
followed by a few days  of  decline before
sMMO activity was essentially eliminated.
This was  true for both  bench- and pilot-
scale systems.  Day-to-day  treatment effi-
ciency,  however,  changed  considerably,
even though  the growth rate (and thus,
the cell concentration in the culture ves-
sel)  remained constant. This  implicates
the physiological  conditions of the bacte-
rial culture, which are  probably  affected
by fluctuating concentrations of metabolic
byproducts (such as methanol) or  by com-
petition from  other organisms in  the cul-
ture  vessel.
   During  the pilot demonstration, meth-
ane  was used at a rate of 240 ml/min at
standard temperature and  pressure. The
apparent yield based on this flow  rate and
cell production rate was 3 x 10~3 g cells/g
theoretical  oxygen demand. Since meth-
ane  is  a  highly usable substrate for these
 Table 2. Bioreactor Operational Parameters for Bench and Pilot-Scale Systems
                             Units
  i1" i  i   set:

 Volume                       L
 Cell dilution rate                h-r'
 Cell density                   Asfg
 Methane flow rate              L-ftr'
 Air flow rate                   L*hr''

 Plug Flow Contactor:

 Length                       rn
 I.D.'                         cm
 Volume                       L
 Culture medium flow/ (qj         L-hr'
 Groundwatar flow (q,J           L*hr1
 Total bioreactor flow (Qb)        L-hr1
 HRT                         hr
       Bench Unit
         1
        0,02
        6,1
         1.05
       21
        0,61
        2,54
        0.31
        0.0280
        0.252
        0.280
        1.1
Pilot Unit
 300
   0.02
   1.8
  14.4
 30
   5
 80
   6
 SO
 66
   0.91
organisms and since typical yields on car-
bon substrates are >10'' g cells/g BOD,  a
high degree of methane stripping was sus-
pected. Based on methane cost of $0.507
hundred ft3, the calculated cost of meth-
ane  during  the  pilot demonstration  was
approximately  $0.33/1000  gal of  water
treated.
  Although  methane was added via the
gas transfer device, air was still introduced
through sparging devices within the cul-
ture vessel. The efficiency of methane use
would  probably  be  improved by adding
both air and methane through the gas
transfer device and,  thus, sparging would
be avoided. In  addition, during  this  test,
the vessel was  stirred by an  agitator that
could be avoided  or at least minimized.
These  improvements would  increase the
efficiency of methane  use and,  thus, re-
duce the  cost  associated with  methane
supply.  (Theoretically,  minimum rates of
bacterial methane  consumption would re-
sult in  methane costs of < $0.05/1000 gal
of treated water.) Thus, it is expected that
using this technology would result in lower
treatment costs than  would either  ad-
vanced oxidation  or  carbon  adsorption/
disposal.

Conclusions  and
Recommendations
   It was concluded that:
  . methanotrophic   TCE  degradation  in
    this  two-stage   bioreactor system  is
    feasible, and
  . the cost of methane necessary  to
    support TCE  biodegradation  is  not
    excessive in relation to the costs of
    other  technologies available  for
    destructive TCE removal  from water.
  The  extent of degradation of TCE from
day to day was highly variable. Conversely,
the culture  xlfinsijv jf^A was  relatively
stable at approximately 1.8. Although the
culture was not axenic, M.t. OB3b  con-
centrations in the culture medium remained
high through the  end  of the  test  runs.
Thus, the fluctuations in TCE degradation
activity more likely resulted from changes
in the expression of sMMO by the culture.
Future studies should focus on stabiliza-
tion of the culture for consistent, long-term
treatment of chlorinated, volatile, aliphatic
hydrocarbons.

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                                                                                             \Uathanm
                                                                Effluent
Figure 1.   Schematic diagram of the bioreactor system. In the bench-scale system methane and air were introduced to the culture vessel through a glass
           air diffuser.  The diagram includes a representation of the bubbleless gas-saturating device ("Methane Exchg") that was used in the pilot-scale
           system.

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                      I
                      LU
                               600
                               500
                               400
                               300
                               200
                                100
                                                                                                       n  INFL
                                                                                                       O  EFFL
                                    0123
                                                                HRT (hours)
Figure 2.  Bench-scale TCE concentrations in plug-flow reactor influent and effluent streams.  Hour zero corresponds to the first TCE measurement
          after the flow of bacterial culture to the reactor began.
                              1500
                              1000
                      O
                      Ul
                               500
                                 0
                                              10
20
50
 Figure 3.
                                                        30
                                                      HRT (mm)
TCE concentrations at various distances down the length of the plug-flow reactor as a function of the hydraulic residence time to thatport. Results
are from the third day of operation of the pilot reactor, showing approximation of reactor performance to first-order kinetics.   The parameters
(estimated by nonlinear regression ± S. E. of the estimate) were Sg =  1896± 40 ppb and K, = 3.43 ± 0.14 x HWmin (2.06 ± 0.08/hr).
concentration in the plug flow reactor was 3.6 mg dry weight/L.

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