II al , ' r Rt.war(h Vol. 13. pp. 485 to 4.92 IX)43-1354 7 t) 0601-(klgSS(12.00 1) ( Pe rgamo n Press Lid 1979 Pr in ted in Grea l Bri tain

BIOASSAY F O R M O N I T O R I N G B I O C H E M I C A L M E T H A N E P O T E N T I A L AND ANAEROBIC TOXICITY

W. F. OWEN,* D. C. STUCKEV, J. B. HEALV, JR., L. Y. YOUNG and P. L. McCAgrV

Department of Civil Engineering. Stanford University, Stanford, CA 94305, U.S.A.

(Received in reri.~ed form 19 December 1978)

Abstract--Techniques are presented for measuring the biodegradability (Biochemical Methane Poten- tial--BMP) and toxicity (Anaerobic Toxicity Assay--ATA) of material subjected to anaerobic treatment. These relatively simple bioassays can be conducted in most research laboratories without the need for sophisticated equipment. BMP is a measure of substrate biodegradability determined by monitoring cumulative methane production from a sample which is anaerobically incubated in a chemically defined medium. The ATA measures the adverse effect of a compound on the rate of the total gas production from an easily-utilized, methanogenic substrate. These techniques are demonstrated by an analysis • of the BMP and ATA of processed samples of peat.

INTRODUCTION Bioassay techniques for measuring the presence or

A simple and inexpensive procedure is needed to absence of inhibitory substances offer the most pro- monitor relative biodegradability and possible toxi- mise for resolving anaerobic treatment problems city of constituents in feed sources to anaerobic treat- because they are relatively simple and inexpensive, merit processes. In order to satisfy these needs, a and do not require knowledge of specific inhibitory batch anaerobic bioassay technique was developed, substances. Also, bioassay techniques are essential for The general procedure can be modified to estimate determining biodegradability since no chemical pro- either the Biochemical Methane Potential (BMP) or cedure is available which distinguishes between biode- to provide an anaerobic toxicity assay (ATA). For gradable and non-biodegradable organics. illustration, specially processed samples of peat were Both continuous (or semi-continuous) and batch- analyzed by these two procedures and the results are feed techniques have been used to evaluate toxicity presented to demonstrate the usefulness of the tech- and biodegradability. The continuous procedures niques, closely simulate full-scale anaerobic operation; how-

BACgGnOUND ever, they are costly in terms of facilities, equipment,

Anaerobic treatment processes are widely used for time, and personnel. Batch bioassay techniques do biological stabilization of concentrated organic not have these limitations and thus permit the evalu- sludges. They provide a significant advantage over ation of a wide range of variables. Batch techniques aerobic processes since they produce more energy in can evaluate the influence of shock loads, but, in gen-

eral, do not simulate the effects of real systems as the form of methane gas than is required for oper- well. However, they are still very useful for sorting ation. Because of dwindling reserves of fossil fuels,

recent interest has grown in the potential of using out important variables and for the development of anaerobic processes for converting organic residues an efficient continuous-feed assay program.

The Warburg respirometer has been widely used into methane gas, an easily transported, clean-burning fuel. However, doubts have been cast on anaerobic as a batch procedure to evaluate biodegradability and

toxicity in aerobic systems. It has also been adapted treatment efficiency and process reliability since many potential residues for bioconversion are relatively for similar analyses with respect to anaerobic treat- non-biodegradable, and also may contain materials ment processes (McCarty et al., 1963; Kugelman & which are toxic to methanogenic micro-organisms. McCarty, 1965 and Gossett & McCarty, 1976). How-

In the past, the cause of anaerobic system failures ever, the Warburg respirometer has sev.eral limi- has been difficult to assess because of the complex tations: (1) it is costly and requires some degree of mixtures being treated. Other difficulties include skill to operate, (2) a given instrument is limited in

the number of samples that can be analyzed at one analysis for the great variety of potential inhibitors (many of which are not yet identified), and the lack time, (3) sample size is limited, making subsequent of understanding of the interactions between inhibi- analyses difficult, (4) it is difficult to sample the gas

and liquid phases during the assay, and (5) extended tors, other constituents in the digesting mixture, and incubation times are impractical and produce incon- the methanogenic bacteria. It has also been difficult sistent results. to distinguish between failures due to toxic materials

In order to circumvent most of the above difficul- and those due to improper design or operation. ties, the theory and procedures developed for the

• Present address: Culp/Wesner/Culp, P.O. Box 40, El anaerobic Warburg were combined with serum-bottle' Dorado Hills, CA 95630. techniques for cultivation of anaerobes as described

485

486 W.F. OWEN, D. C. STUCKEY, J. B. HEALY, L. Y. YOUNG and P. L. MCCARTY

Table 1. Stock solutions for preparation of defined media

Solution Compound Concentration (g J - 1 )

SI Sample <2El -~ degradable COD* in assay liquid (estimated)

$2 Resazurin I

$3 (NH,),HPO4 26.7

S'~ CaClz "2H20 16.7 NH4CI 26.6 MgCI: "6H,O 120 KCI 86.7 MnCI2 '4H20 1.33 CoOl, "6H20 2 H~BO3 0.38 CuCIz'2H20 0.18 NazMoOo" 2H20 0.17 ZnCI2 0.14

$5 FeCI,-4H20 370

$6 Na_,S. 9H_,O 500

$7 Biotin 0.002 Folic acid 0.002 Pyridoxine hydrochloride 0.01 Riboflavin 0.005 Thiamin 0.005 Nicotinic acid 0.005 Pantothenic acid 0.005 Bt2 0.0001 p-aminobenzoic acid 0.005 Thioctic acid 0.005

* Chemical oxygen demand.

by Miller & Wolin (1974). Anaerobic serum bottles ($2) is added to detect oxygen contamination (pink containing samples, defined media, and seed inocula when oxidized), and sodium sulfide ($5) is added to are incubated at the desired temperature, and respect- provide a reducing environment. The procedure for ire gas productions are monitored volumetrically preparing 1.8 I of defined media is given in Table 2. using the syringe method of Nottingham & Hungate The final assay concentrations of nitrogen, phos- (1969). The liquid and gas phases can be sampled phorus, and alkalinity are, respectively: 122mgl - t as periodically by syringe extraction for subsequent ana- N, 19 mg 1- t as P, and 2.500 mg 1- t as CaCO,.

lyses. Anaerobic transfers GENERAL ANAEROBIC BIOASSAY

The defined media is equilibrated to assay tempera- TECHNIQUES

ture, inoculated, and transferred into serum bottles. Preparation of assay bottles For the BMP assay, inoculation is accomplished

The BMP assay is conducted with Coming No. anaerobically by inserting a gas flushing needle into 1460, 250 ml reagent bottles (264 + 1 ml actual the neck of the media flask while adding 200 ml of volume with Bittner size No. 16 serum cap in place), seed organisms to 1,800ml of defined media. A 20~:0 and the ATA with 125 ml reagent bottles with rubber by volume inoculum is used, and in certain cases, serum caps of appropriate size. Bottles are gassed at defined media and seed inocula can be tranferred sep- a flow rate of approximately 0.5 1 min - t for 15rain arately into serum bottles. with a mixture of 30% CO2 and 70% N2, then stop- The anaerobic transfer of defined media is depicted pered and equilibrated at incubation temperature in Fig. 1. Assay bottles, gas tubing, and transfer #ass- prior to introducing samples, defined media, and in- ware are initially gassed, and samples are added to ocula, bottles and equilibrated to the incubation tempera-

ture. Media is anaerobically pipetted by opening and Defined media closing appropriate pinch valves (V1 and V2), while

f Concentrated stock solutions (Table 1) are used for flushing the assay bottle and media flask at 0.51

preparing the defined media and are stored at 4°C. rain - t with 30:70 volume ratio of CO2:N2. When The defined media contains nutrients and vitamins the assay bottle is filled to the approroriate volume, for mixed anaerobic cultures as adopted from studies a serum cap is inserted while simultaneously remov. by Speece & McCarty (1964) using additional vita- ing the gas flushing needle. After equilibration for one rains as indicated by Wolin er al. (1963). Resazurin hour at the incubation temperature, gas volumes are

Bioassay for monitoring biochemical methane 487

Table 2. Preparation of defined media*

Step Instruction

1 Add 1 liter of deionized water to 2 liter volumetric flask. 2 Add the following:

1.8 ml $2 5.4 ml $3 27 ml $4

3 Add deionized water up to 1,800 ml mark. 4 Boil for 15 rain while flushing with N 2 gas at approximately 1 l min- ' . 5 Cool to room temperature (continue flushing with N 2 gas). 6 Add the following:

18 ml 57 1.8 ml 55 1.8 ml 56

7 Change gas to 30% CO2, 70% N 2 mixture and continue flushing at 1 Imin- i . 8 Add 8.40 g NaHCOs as powder. 9 Bubble 30% CO2:70% N 2 gas mixture through porous diffuser until media pH stabilizes at approximately

7.1. 10 Carefully seal volumetric flask while minimizing introduction of air into container.

* Media is prepared in a 2 liter volumetric flask marked at the 1,800 ml leyel. It can be stored indefinitely provided oxygen is excluded. Seed organisms are added to the media just prior to transfer by filling the volumetric flask to the 2 flier mark with seed (BMP only).

"zeroed" (ambient pressure) with a syringe and the original equilibration volume. In order to continue bottles are ready for incubation and sampling, the assay, g ~ can be reinjeeted into respective bottles

without contaminat ion or loss at this point, or the Gas measurement syringe full of gas can be removed for wasting.

Gas-volume sampling and removal during incuba- t ion is performed with glass syringes (5-50 ml depend- BIOCHEMICAL METHANE POTENTIAL ing on gas volume) equipped with 20-gauge needles. (BMP) The sample syringe is initially flushed with the Biochemical methane potential is a measure of CO2:N2 gas mixture and lubricated with deionized sample biodegradability. The methane contribution water. Readings are taken at the incubation tempera- resulting from sample decomposition is determined ture and the syringe is held horizontal for measure- by subtracting background values, obtained from ment. Volume determinations are made by allowing seed-blanks, from the sample totals. Seed-blanks are the syringe plunger to move (gently twirling to pro- prepared according to the prescribed procedure with- vide freedom of movement) and equilibrate between out addition of an organic substrate. Samples are the bottle and atmospheric pressures. Readings are anaerobically added to the 250mi serum bottles verified by drawing the plunger past the equilibrium before the transfer of inoculated defined media and point and releasing; the plunger should return to the duplicates are prepared for all samples.

i I

~v. co~ Graduated ~ _ ~ , ~ ~o I00 mt ! volumetric o ] / ~pqrt t

Defined media " Flushing

Mognetic Serum Water stirrer bott le seal

Fig. 1. Schematic diagram of procedure for the anaerobic transfer of defined media into serum bottles.

488 W.F. OWEN, D. C. SrUCKEY, J. B. HEALY, L. Y. YOUNG and P. L. McCARrY

Proper sample size and liquid-to-void volume ratio percent organic matter converted to methane using are important for the precision and accuracy of the theoretical 0.350m 3 CH,~ at STP produced per results, and are chosen with the following guide-lines: kilogram COD converted (McCarty, 1964). (11 provide a measurable, but not excessive, amount of methane, usually 20-120ml, (2) ensure that .ANAEROBIC TOXICITY ASSAY

(ATA) nutrients will not be limiting, and (3) eliminate poss- ible substrate toxicity. For a readily-degradable and Anaerobic toxicity is determined as the adverse non-toxic organic, such as pure cellulose, a 2-20 ml effect of a substance on the predominant meth- liquid sample (or a dry sample) containing 150rag anogens, i.e., those that convert acetic and propionic chemical oxygen demand (COD) is generally used acids to methane (Jeris & McCarty, 1965). Assay-bot- with a final total liquid volume in the assay bottle ties are prepared similar to the BMP assay with of 160 ml (void-volume equals 104 ml). The estimated defined media, seed inocula, and samples. In addition, degradable COD is kept to less than 2 g 1- 1 in the a "'spike" containing acetate and propionate is added assay liquid. These ratios can be adjusted when to each bottle. The metabolism rate of the acetate- toxicity and/or low degradability are anticipated, propionate spike is monitored by total gas production and multiple dilutions can be employed when an esti- using the syringe method. Inhibition due to sample mate of degradability is not available. Total liquid addition is determined as a decreased rate of gas pro- volumes up to 200 ml can be used in order to decrease duction relative to an active control. The ATA is typi- the void-volume and improve the accuracy of meth- cally conducted under quiescent incubation condi- ane determinations when low gas production is tions. However, certain cases may warrant agitation expected, since virtually all of the solids and micro-organisms

A detailed methane balance is kept for each sample settle and are concentrated at the bottom of assay during the assay, and gas volumes are monitored bottles when not stirred, Therefore, agitation may be periodically. Gas is wasted as necessary to prevent desirable when toxicity is associated with particulates, inadvertant gas leakage due to excessive pressure, either directly or by adsorption. which is generally indicated when the difference To test a substance, assay concentrations are between internal and atmospheric pressures is greater selected to provide a range from non-inhibitory to than 0.5 atm. Methane content is determined severely toxic. In general, five to ten assay concert- whenever gas is removed. Gas samples to be analyzed trations are selected for a substance along with three by gas chromatography are extracted from serum bot- controls. Serum bottles are prepared and equilibrated ties with a precision gas-syringe when the assay is to assay conditions, samples are anaerobically added, near atmospheric pressure, i.e. just after wasting. At and defined media is anaerobically transferred to the the termination of the test period, the quantity of bottles. methane within the assay volume is added to that For concentrated toxic substances (i.e., when wasted during incubation and sampling and the sample volumes represent less than 100~,~ of the total resulting total methane production is used for calcu- assay liquid), all bottles are prepared identically by lating BMP. filling to 48.0 ml with inoculated media (5.38 g l-

Test precision and accuracy are functions of the sodium bicarbonatei and the sample. For this case, standard error of estimate of the methane contribu- media is inoculated prior to its transfer using 2000 tions due to seed inocula and substrate metabolism, by volume of seed inocula. When sample volumes are In order to minimize the methane contribution from greater than 5 ml (10°o of assay liquid), varied seed organisms, a semicontinuous seed digester is volumes of defined media are employed in order to maintained with a low organic loading, and seed for maintain the total volume constant at 38.0ml prior inocula is removed from this digester just prior to to addition of 10ml of inocula (added separately to normal feeding. The total methane produced from the each bottle). seed can be limited to less than 1.5 + 0.5 ml over a Finally, all bottles are fed 2.0ml of acetate-pro- 30-day incubation period. A typical sample can corre- pionate solution containing 75 mg acetate and spondingty be analyzed as 50.0 + 1.0 ml of methane 26.5 mg propionate by syringe injection, resulting in during the same period resulting in an overall preci- a 50.0 ml liquid assay volume. After equilibration and sion of _+200. zeroing the assay is commenced.

The incubation period is typically 30 days which ~- The first week of incubation is critical. Gas produc- ensures virtually complete decomposition of b iode - ' tion is measured once or twice daily for the first seven gradable organics in most cases. This procedure elim- days and periodically thereafter. Gas is expelled after inates variations due to differing metabolism rates, each measurement. Some organics, however, may require a longer period Total gas production data are employed for deter- for acclimation, mining relative rates of metabolism of the acetate-

BMP is referenced either to sample volume (m 3 propionate spike among samples. The maximum rate CH4/m 3 sample), sample mass (m 3 CH,,/kg sample), of gas production is computed for each sample over or sample organic content (m 3 CH4/kg COD). The the same time period and data are normalized by latter method permits direct transfer of results into computing ratios between respective rates for samples

Bioassay for monitoring biochemical methane 489

and the average of the controls. This ratio is desig- ioo (o)Corrrmt ~/NaOH hated the maximum rate ratio (MRR). Since measure- ment of gas production is relatively accurate, a MRR ac ~ ~ of less than 0.95 suggests possible inhibition, and one - - " less than 0.9 suggests significant inhibition. However, 60 data interpretation is sometimes complicated by gas production resulting from sample decomposition, and 4o also by varying ratios of carbon dioxide and methane production. Nonetheless, relative toxicity can be 20 ~ [ t ~ S ~ d c o t r t r o l assessed reasonably well in most cases, and, if deemed E o . ~ r ~ ~ . , , , t t i ~ t L J necessary, can be confirmed by semi-continuous - studies, g 14o

~ (b) Heot-'trgot"ed ot IO0"C ~

EXAMPLE APPLICATION OF BIOASSAY J20 ~ . o ~ T E C H N I Q U E S /

O IOO The use of BMP and ATA are demonstrated with 4 4

data from analysis of thermochemically pretreated / ~ j peat.* The peat samples were selected from a separ- so ate, ongoing study to illustrate many important con- siderations in application of the anaerobic bioassay 6o methods presented. 4o

The peat samples analyzed had previously been /~ x tFed control) ~. . - - - - alkaline heat treated for one hour at 100, 200, and 2o f f ~ 250°C in an attempt to increase anaerobic biodegra- dability. The samples were analyzed by BMP and I I I I I I I ATA procedures and results are "compared here to o 4 e ~z J6 2o 24 26 s an untreated control (P1) and an alkali-treated con- Tim=, daws trol that was not heat treated (P2). The sample C O D Fig. 2. Results of anaerobic toxicity assays for controls values are listed in Table 3. (P2) and heat treatment at 100C (P3t.

Anaerobic toxicity assay (A TA) ter fed waste activated sludge at a 15-day solids reten- tion time. Finally, 2.0ml of the acetate-propionate

The various peat samples were evaluated at total spike was added by syringe. solids concentrations of 0.4, 2.0, 4.0, and 16.0 g i - 1. In addition to the standard "spiked" or fed control. The assay was conducted at 35~C using a total liquid an "unspiked" seed control was also observed. The volume in each bottle of 50 mL Sample volumes rang- resulting data are summarized in Figs. 2 and 3. The ing from 0-20ml were mixed with defined media data for intermediate concentrations (2.0 and (5.38gl - I NaHCOa) to give a combined volume of 4.0g1-1) of P2 and P3 were omitted for clarity, as 38.0ml. A seed inoculum (10ml) was transferred to they fell between the extremes. Also, the non-alkali- each assay from a mesophiiic (35~C) laboratory diges- treated peat control (P1) is not shown since assay

results were the same as for the alkali-treated control * Peat from Minnesota supplied by Dynatech R/D Com-

pany, Cambridge, Mass. (P2).

Table 3. Chemical oxygen demand of alkaline heat-treated peat*

Pretreatment Sample COD,§ Sampler temperature, + "C g l- 1

Pl Peat control 55.3 P2 Alkali-treated control 55.3 P3 100 49.5 P4 200 49.5 P5 250 51.6

* Peat-Slurry initial conditions §

Total solids = 39.9 4- 0.3 g I-1 Volatile solids = 36.6 + 0.2 g l - ~ t91% of TS) COD/VS ratio = 1.51

Total Kjeldahl Nitrogen = 1.2g N/100g VS.

1"Approximate 5 h contact time with 400mequiv I- J sodium hydroxide for all alkali-treated samples, then neutralized with HCI prior to bioassay.

~: Reaction temperature held for l h under nitrogen atmosphere. § All analyses conducted according to standard procedures (APHA, 1976).

w.R. 13~0.-- a

490 W.F. OWEN, D. C. SrucKEY, J. B. HEALY, L. Y. YOUNG and P. L. McCARTY

tOO ,___,_~ _~ ~,,,,,,- __-.--..,,,e~ 3); therefore, cumulat ivegas production data for this ( a ) ~ o , ~ period were used for computing MRRs (Table 4).

so ~ _ Alkaline heat treatment of peat resulted in the pro-

60 ~ d" F ~ t , g/t duction of inhibitory products, especially at high tem- ~fl- / o o.a peratures. With 200°C pretreatment (P4) inhibition

~t a 2.o was noted at both 4.0 and 1 6 . 0 g l - a ( M R R i n T a b l e 4 ) . ,,o E / ~ 4.o

[ • 16.o However, after 6 days, gas production was greater ~ × (Fed conerol ) ....--, with 4.0 g 1 - t (Fig. 3a) than with the fed control. With

20 . . . . . 16.0g 1- ' gas production was severely inhibited for eod control

o r~ I u I i i r I 13.5 days, al~er which time acclimation OCCUlTed and cumulative gas product ion soon exceeded the control.

~ , o o With 2.50~C pretreatment, inhibition was noted at l o ) H _ _ ~ a ~ . - ~ 2.0, 4.0, and 16.0g 1-1 as determined by the MRR

so values (Table 4). In the first two c,ases, acclimation occurred rapidly; but with 16.0gl - t only partial

60 / * acclimation o c c u r r e d after about 20 days. It is not

. , , - ¢ whether acclimation due destruction apparent was to 40 ~ _ _ . , ~ r / of toxin, enzyme adaptation, or selection of more re-

zo ~ ~ sistant organisms. The precision of the ATA procedure is illustrated

for the cases where replicate samples were used 0 4 8 12 16 20 24 28 32 (Table 4). In general variations in gas production and

Time, cloys MRR were less than 3~o. Fig. 3. Results of anaerobic toxicity assays for heat treat-

ment at 200'~C (P4) and 250°C (P5). Biochemical methane potential (BMP)

BMP assays were conducted at 35°C and seed Maximum substrate utilization generally occurred organisms were obtained from a mesophilic (35°C)

during the first five days of incubation (Figs. 2 and digester fed alkaline heat-treated newsprint (largely

Table 4. Cumulative gas production and MRR for anaerobic toxicity assays (ATA)

Cumulative gas production (ml) Peat

concentration Incubation time: Sample (g 1- t ) 5 days 30/days 45½ days CH,/CO~ MRR't

Fed Control - - 59.1 + 1.7 76.7 82.8 + 2.3 2.0 - - (n = 3)

Seed Control - - 9.0 + 0.2 28.9 32.9 _ 2.4 - - - - (n = 3l

PI Peat control 0.4 58.4 74.5 79.8 2.0 0.99 no NaOH 2.0 59.7 76.4 78.3 2.0 1.01

4.0 61.1 77.0 82.0 2.0 1.03 16.0 62.0 81.2 86.5 1.8 1.05

P2 Peat control 0.4 58.5 74.6 80.0 2.0 0.99 + NaOH 2.0 59.4 75.7 81.7 2.0 1.01

4.0 60.5 76.5 83.5 2.0 1.02 16.0 52.6 82.8 88.8 2.0 0.89

P3 100cC + NaOH 0.4 60.2 76.5 82.6 2.0 1.02 (n = 5) 2.0 62.7 + 0.4 79.7 + 0.3 86.7 + 0.3 2.0 1.06 _ 0.03

4.0 66.3 86.7 94.3 2.0 1.12 16.0 62.5 131 138 2.0 1.06

P4 200~C + NaOH 0.4 58.9 74.8 79.5 2.0 1.00 2.0 61.0 80.8 83.6 2.1 t.03 4.0 54.8 93.0 97.4 2.2 0.93

16.0 17.4 96.8 109 2.0 0.29

P5 250c'C + NaOH 0.4 57.5 73.5 78.0 2.0 0.97 (n = 5) 2.0 53.7 _ 0.9 80.4 4- 0.3 82.3 __+ 0.2 2.2 0.91 _ 0.03

4.0 37.6 88.8 93.0 2.3 0.64 16.0 11.3 55.0 86.0 2.4 0.19

* Methane-to-carbon dioxide ratio within the assay, measured after 45 days. Sample gas productio'n (sl~ik~ . . . . .

"t" MRR = Maximum Rate Ratio = =-'--r--. ~ , measured at 5 days.

Bioassay for monitoring biochemical methane 491

Table 5. Anaerobic biodegradability of alkaline heat-treated peat

Treatment Assay* Conversion:~ temperature concentration B M Pt" efficiency

Sample °C g I-1 m 3 CH4/kg COD m 3 CHJkg TS

Pl Peat control 0.5 0.000 0.000 0 Pl Peat control 2.0 0.000 0.000 0

P2 Alkali-treated 0.5 0.007 0.010 2 control

P2 Alkali-treated 2.0 0.006 0.090 2 control

P3 100 0.5 0.035 0.043 10 P3 1O0 2.0 0.033 0.041 9

P4 200 0.5 0.071 0.088 20 P4 200 2.0 0.074 0.090 21

P5 250 0.5 0.089 0.116 26 P5 250 2.0 0.078 0.101 22

* Total solids concentration of dry peat sample within assay liquid. ? Biochemical Methane Potential, at STP, incubation time = 31 days; referenced to sample COD and total solids

('IS}. Conversion etficiency of organics to methane; based on a theoretical BMP of 0.350m 3 CH,/kg COD converted

at STP.

iignin, cellulose, and hemicellulose) at a solids reten- be partially converted to methane gas by anaerobic tion time of 15 days. These organisms had been culti- treatment. The maximum conversion efficiency of rated on the same substrate for 1.5 yr, and therefore 26% represents 0.125 m 3 of methane at STP per kg were acclimated to most of the products likely to be of peat (dry solids basis assuming no losses during formed by alkaline heat treatment of peat (largely lig- pretreatment) or approximately 4,650 kJ in methane nin). Use of acclimated organisms eliminated the need production per kg of peat pretreated and digested. for extended incubation periods. These results are tempered by the formation of in-

BMP assays were conducted at two concentrations hibitory products during heat treatment. The data in of peat within the assay bottle after dilution (0.5 and Table 4 indicate that slight inhibition of methane for- 2.0 g 1-1 of total solids) to confirm that methane pro- mation occurred for P4 (200°C) at 4.0 g I- 1 and P5 duction was not inhibited by toxic materials (BMP (250°C) at 2.0g I - 1 and that significant inhibition values should be identical and results can be con- occurred for higher concentrations of these products. firmed by ATA). A total liquid volume of 200 ml was A methanogenic population, not previously accli- employed so that the expected low methane produc- mated to heat-treatment products, was able to par- tion could be accurately measured, tially acclimate to the highest concentrations tested

Anaerobic biodegradability results are detailed in (16g 1-t) of both P4 and P5: it took approximately Table 5. Untreated peat was not digestible and pro- twice as long for acclimation to P5. duced no methane. The digestibility of alkaline pre- In conclusion, the al/aerobic bioassay techniques treated peat increased with increasing treatment tern- described are relatively rapid and accurate methods perature up to the maximum temperature evaluated, for assessing methane potential and toxicity of 250°C. The maximum conversion efficiency was 26% samples. Several variables can be investigated and the observed at 250°C. For these pretreatment conditions, more promising conditions screened for more detailed sfight inhibition was noted with 2.0 g 1-1 samples studies. Furthermore, results can be expressed in use- (BMP for both sample concentrations differ, Table 5). ful engineering terms which can be applied for assess- The ATA results indicated that the 0.5 g !-1 concen- ing process performance. The procedures are quite tration of this sample was, not inhibitory; thus, a flexible allowing fundamental studies beyond those BMP of 0.089 m 3 CH4 kg- t COD represents the non- demonstrated. For example, both liquid and gas inhibited bioconversion efficiency for P5. No inhibi- phases of the assays can be monitored. In this way, tion was noted in any other BMP assays, the progress of substrate utilization and intermediate

formation and utilization can be monitored Simul- DISCUSSION AND SUMMARY taneously. These possibilities are important consider-

It is useful to summarize the results from pretreated ations for identifying the cause and effect of toxicity. peat samples in order to demonstrate the utility of the anaerobic bioassay techniques presented. Aclmowledoements--This research was supported by the

Increased pretreatment temperature for alkaline Minnesota Gas Company, Minneapolis, Minnesota, and peat increased both the degradability and toxicity of by Dynatech R/D Co., Cambridge, Massachusetts. The

bioassay procedures were developed under Grant No. the products. Alkaline heat treatment converted peat DOE-EY.76.S-03-0326-PA-4,4 from the U.S. Department from a non~ligestible organic into a product that can of Energy.

492 W.F. OWEN, D. C. SrucnEY. J. B. HE^L¥, L. Y, Yot;Nc; and P. L. McCAarY

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Gossett J. M. & McCarty P. L. (1976) Heat treatment of Miller T. L. & Wolin M. J. (1974) A serum bottle modifica- refuse for increasing anaerobic biodegradability. In Bio- tion of the Hungate technique for cultivating obligate chemical Engineerinq-Eneryy, Renewable Re.sources and anaerobes. Appl. Microbiol. 27, 985-987. New Foods, Vol. 72 No. 158, pp. 64--71. American Insti- Nottingham P. M. & Hungate R. E. (1969) Methanogenic tute of Chemical Engineers Symposium Series. fermentation of benzoate. J. Bact. 98, 1170-1172.

Jeris J. S. & MeCarty P. L. (1965) The biochemistry of Speece R. E. & McCarty P. L. (1964) Nutrient require- methane fermentation using C ~4 tracers. J. War. Pollut. ments and biological solids accumulation in anaerobic Control Fed. 37, 178-192. digestion. Adrances in Water Pollution Research, Vol.

Kugelman I. J. & McCarty P. L. (1965) Cation toxicity 2, p. 305. Pergamon Press, London. and stimulation in anaerobic waste treatment. J. Wat. Wolin E. A., Wolin M. J. & Wolfe H. S, (1963) Formation Pollut. Control Fed. 37, 97. of methane by bacterial extracts. J, biol. Chem. 238.

McCarty P. L. (1964) Anaerobic Waste Treatment Funda- 2882-2886.