BIOTECHNOLOGY AND BIOENGINEERING, VOL. XVII, PAGES 1634-1662 (1975)

Thermophilic Microbiological Treatment of High Strength Wastewaters with Simultaneous Recovery of Single Cell Protein G. A. SURUCU,* R. S. ENGELBRECHT, and E. S. K. CHIAN, Department of Civil Engineering, University of Illinois, Urbana, Illinois 61801

Summary Simultaneous removal of organic materials and recovery of protein in the form of bacterial cells from a simulated high strength biodegradable wastewater was studied using thermophilic aerobic microorganisms. A naturally occurring mixed culture of thermophilic microorganisms was obtained from soil, wastewater, hay, silage, etc. A chemically defined medium containing glucose along with other essential nutrients was employed as the feed. The kinetic behavior of the culture was studied in a continuous culture a t an optimum temperature of 58°C. Studies were also performed on the effects of solids retention time (SRT) on the observed cell yield and the protein and ash content of the harvested biomass. An economic analysis of the process for single cell protein recovery was given.

INTRODUCTION A growing concern for acute food shortages has led to the examination of a variety of sources as potential supplements to the world's food supply. Among these, the consumption of biomass of microorganisms as a protein and vitamin source probably represents the best opportunity for the development of a unique nonagriculturally based food supply. This is mainly due to the rate of protein product,ion as well as nutritional values being very much in favor of single cell protein (SCP). Recently, it has attracted the interest of many investigators to redeem SCP from inexpensive carbon sources especially from wastes.' Bagasse, spent sulfite liquor, cow manure, hydrocarbons, etc. have been used for the production of SCP.2-4 It is conceivable that the utility of any process depends upon economic considerations. The important factors associated with the * Present address: Middle East Technical University, Department of Environmental Engineering, Ankara, Turkey. 1639 @ 1975 by John Wiley & Sons, Inc.

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SURUCU, ENGELBRECHT, AN11 CHIAN

economics of production of microbial protein are : availability and cost of raw material (carbon source), sterility requirements, fermentation process consideration, i.e., residence time in reactor, operation temperature, cooling water requirements, oxygen requirement, cell yield, cell recovery, and product value. All of these factors, except the one related to cell yield, suggest, that recovery of protein in the form of bacterial cells from the thermophilic aerobic treatment of high organic strength wastewaters should receive strong considerations. Several reasons support this. 1) Some industries, such as dairy or cheese making plants or starch or corn processing factories, fermentation industries, etc. generate a large quantity of waste organic materials in rather hot and concentrated forms. These wastes are biodegradable and provide practically zero cost carbon sources for thermophilic SCP production. 2) Sterility requirements during the production of SCP a t mesophilic temperatures are usually quite stringent. However, it may not be necessary t o work under aseptic conditions in the thermophilic range since the high temperature (55-65°C) of the system will prevent the growth of many unwanted contaminants, especially the pathogens t o humans and animals. However, contamination by thermophilic bacteriophages may pose problems as many phages can survive beween 65-75°C for several hours.5 3) Higher growth and specific substrate utilization rates at higher temperatures have been frequently reported in the literature. Numerous authors6-'* have found higher reaction rates a t elevated temperatures. Therefore, with thermophilic conditions, the required residence time in the reactor will be less than that required with mesophilic conditions. 4) In industrial fermentations and also in production of SCP a t mesophilic conditions, cooling is frequently necessary to remove heat produced by microbial t h e r m o g e n e ~ i s . ~Use ~ ' ~ of a thermophilic system may solve the problem of cooling. It has also been shown that a thermophilic system for treating wastewaters can operate by sustaining itself at the temperature range between 50-60°C with the heat produced by therm~gcnesis.'~*'~ Therefore, thermophilic operation of the system may not require an additional external heat source. 5) Although the saturation value of oxygen a t 60°C is less than that at 35°C (8.3 mg/liter a t 25°C versus 4.6 mg/liter a t 60"C), the mass transfer coefficient of oxygen a t higher temperatures is increased because of the highcr diffusivity of oxygen in water, e.g., 2..5 X

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sq cm/sec a t 25°C versus 6.1 X sq cm/sec at 6OoC.l5 Therefore, the oxygen transfer rate a t thermophilic conditions should be as good as, and sometimes better than, that a t mesophilic temperature ranges. 6) Finally, studies by Mateles et al.,3 Singleton and Arnelunxen,l6 and Bellamy4 have shown that the protein content from thermophilic microorganisms and their amino acid composition, in terms of nutritional value, may be better than the SCP produced under mesophilic temperatures. Thus, the lower yield coefficient obtained with thermophilic organism^'^ may be off set partially by the advantages of thermophilic production of SCP. I n the case of using a wastewater as substrate, it should be noted that under certain conditions the recovery of protein may serve as a means of offsetting a portion of the total treatment cost. From this brief discussion, i t is evident that the potential of recovering SCP from the thermophilic microbiological treatment of high strength wastewaters should be of great interest in industrial wastewater treatment. However, most of the reported data on the thermophilic production of SCP from waste materials deal with solid waste that are primarily cellulosic materials.4.6JJ1 Little or no information is available on the kinetic and operating parameters of a thermophilic mixed culture system treating wastewater. Although Hajny et a1.l' reported on work with thermophilic mixed cultures, they were using anaerobic systems for the degradation of cellulosic wastes only. I n most cases, production of SCP from cellulosic wastes was carried out in a mesophilic second stage using as substrate the cellulose hydrolysates from the first stage digestion step.17J8 A mixed culture of Alcoligenes jaecalis and Cellulomonas sp. was reported by Han and Srinivasanlg for SCP production on chemically hydrolyzed solid wastes, bagasse. The aerobic mesophiles were, however, employed in their studies. The kinetic and operating parameters of the aforementioned thermophilic systems are either unavailable or incomplete. To operate the process for optimum performance, it is necessary t o know the kinetic coefficients and to understand the effects of the operating parameters on the system's performance. Therefore, it was the object of this research to: a) determine the kinetic coefficients of an aerobic thermophilic mixed culture system; b) demonstrate the effects of solids retention time on substrate utilization, oxygen consumption, observed yield, and protein content of the harvested biomass; and c) assess the economics of the protein recovery process.

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SURUCU, ENGELBRECHT, AND CHIAN

MATERIALS AND METHODS Culture Medium

A chemically defined medium containing glucose as the carbon source, and other minimal nutrients, such as Na+, Ca++, Mg++, Fe+++, NH4+,methionine, riboflavin, and phosphate buffer (pH 7) were developed for this study.14 The formula of the medium is given in Table I. TABLE I Composition of Thermophilic Culture Medium

Constituent Glucose Methionine Riboflavin KHzPO4 N&HPO(

Concentration (mg/liter) 2000 60 01.5 1500 3750

Constituent NaCl CaC4 MgCL FeClz NHdCI

Concentration (mg/liter) 1000 50 50 2 1000

Culture

I n order t o simulate the conditions found in wastewater treatment, a mixed culture of thermophilic organisms was used in this study. The mixed culture, obtained from soil, streamwater, raw wastewater and silage, comprised a mixture of organisms having different morphology and nutritional requirements for growth. Three of the dominating organisms were isolated by employing the aseptic technique. These pure cultures, once established, were studied with respect t o colony appearance, motility, spore formation, gram reactions, and morphological characteristics.

Organism no. 1 When grown at 58"C, this organism consisted of small rods. It occurred in chains of two or more organisms and also as single cells. On complete medium solidified with 1.5% agar, it formed small round colonies. This organism was not motile and it did not form endospores nor aerial hyphae with sporofores. It was nutritionally fastidious. It could be grown in nutrient broth but grew better in complete medium. Gram staining tests indicate that it was grampositive organism. Based upon these observations, it was concluded that this organism was probably Lactobacillus tkermophilus.

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Organism no. 2 Organism no. 2 was an aerobic spore-forming rod, when grown a t 58°C. Unlike organism no. 1, i t did not occur in chains but occurred only as single cells. On complete medium, 1.5y0agar plates, it formed fast spreading star-shaped colonies. It grew well in complete medium and was motile. This organism was a gram-positive rod. Based upon its aerobic requirement and spore-forming characteristic, this organism was judged t o belong t o the genus Bacillus.2o

Organism no. 3 Organism no. 3 also was grown at 58°C. It consisted of discrete long rods and formed terminal endospores. The sporangium was swollen by oval spores. On complete medium, 1.5y0agar plates, its colonies spread a thin layer all over the plate. It was motile and gram-positive. This organism grew well in complete medium. Like organism no. 2, this organism belonged to the genus Bacillus, but probably was a different specie than organism no. 2.20

Measurement of Cell Mass The dry weight of biomass was measured by centrifuging a 100 ml portion of cell suspension at SO00 rpm and 4°C for 10 min. The centrifuge used was a Sorvall RC-2 (Ivan Sorvall Inc., Norwalk, CT). After the cells were separated from the supernatant, they were washed with distilled-demineralized water and dried at 103°C in tared aluminum weighting dishes t o a constant weight.

Chemical Oxygen Demand (COD) Analysis Since the specific Glucostat (Worthington Enzymes, Freehold, N.J.) measurement would not detect the metabolic products of glucose which might accumulate in the medium, the chemical oxygen demand (COD) analysis was employed for measuring the organic concentration in the system. Therefore, all of the coefficients in the kinetic equations were expressed on the basis of COD tests. COD determinations were carried out using the dichromate method as described in Standard Methods.21

Biomass Protein Content Measurements The protein content of the harvested biomass was determined by the Folin-Coicalteau test as described by Lowry e t a1.22 The quantitative Folin-Ciocalteau test has the advantage of being applica-

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SURUCU, ENGELBRECHT, AND CHIAN

ble t o dried materials as well as to solutions. Furthermore, the method is both rapid and sensitive. The amount of protein present in the sample was found from a protein standard curve. The protein standard curve was prepared using the same procedure but with a standard solution of 5X crystalline bovine serum albumin (Sigma Chemical Co., St. Louis, 110.). The use of the Lowry method for protein analysis served the purpose of measuring the relative amount of protein in the cells obtained under various operating conditions. However, no effort was made to correlate the protein content measured by the Lowry test to other methods, e.g., total organic nitrogen test.

EXPERIMENTAL APPARATUS Figure 1 shows a schematic diagram of the single-stage, isothermal, completely mixed continuous flow reaction system which was used throughout this study. Water was maintained a t 59 f 0.3"C in the water bath (Lab-Line Instruments, Inc., Melrose Park, Ill.) from which i t was pumped through a stainless steel coil within the reactor. By this arrangement, the temperature of the culture medium could be maintained a t 58 A 0.5"C. Compressed air was moisturized by passing it through a water bottle placed in the water bath. The dissolved oxygen concentration in the reactor was maintained above 2 mg/liter by means of a rotameter and a mixer (T-line lab mixer, Talboys Engineering Corp., Emerson, N.J.). The latter was also employed to keep the reactor completely mixed. The medium was

Fig. 1. Schematic diagram of continuous flow apparatus.

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sterilized to prevent any growth in the storage reservoir during feeding. By adjusting the feed inflow rate, the desired solids retention time (SRT)was obtained. Since no solids recycle was employed, the SRT was essentially equal to the hydraulic residence time. To achieve a constant inflow rate with the peristaltic pump (Model 7014 tubing pump, Cole Parmer Instr. Co., Chicago, Ill.), the electrical power was controlled by a voltage regulator in order to eliminate any fluctuation in the electrical supply. In addition, the tubing was changed on a regular basis.

EXPERIMENTAL DESIGN To determine the kinetic behavior of the thermophilic heterogeneous microbial population and the effects of solids retention time (SRT) on the system characteristics, such as apparent cell yield, substrate utilization, and the protein content of the biomass, a series of experiments were performed at eight different SRT values using the medium given in Table I. Rilateles and ChianZ3have shown that changes in predominance of the mixed microbial population may occur with different dilution rates which, in turn, may influence the results of the study. Consequently, qualitative observations of the culture color and morphology were made throughout the study. A homogeneous mixture of all three heterogeneous organisms described previously with no obvious predominance of population was observed throughout the runs. Also, it was not possible to detect any change in the data obtained a t steady state which could be attributed to a shift in the culture predominance. Therefore, all data were considered in the same manner. To determine the evaporation losses of the system, the effluent was collected for comparison with the volume of feed pumped during the same period, using the one day SRT experiment at 58°C. The volume difference attributed to evaporation losses was negligible and accounted for less than 0.2% loss over the 34 hr test period. The COD test was used to measure the substrate concentration throughout this study. It is appropriate to use the COD test when the substrate is heterogeneous in nature. COD and solids determinations were made daily. It was observed that, for each of the different SRT values, approximately ten volume turnover times were necessary for the systems to reach a steady-state condition as determined by COD and gravimetric cell concentration measurements (Figs. 2 and 3).

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SURUCU, ENGELBRECHT, AND CHAIN

DAYS OF OPERATION

Fig. 2. Two days SRT experiment: ( - - A - - ) biomass concentration; (-0-) effluent COD.

KINETIC MODEL The kinetic model employed was the familiar one first given by 1\.I0nod~~ and Novick and Szilard,Z5and later discussed by Herbert,26 then modified t o include cell decay as advocated by Van UdenZ7and Lawrence and McCarty.28 This model is applicable to systems either

THERMOPHILIC MICROBIOLOGICAL TREATMENT 140

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DAYS OF OPERATION

Fig. 3. Ten days SRT experiment: ( - - A - -) biomass concentration; (0-0) effluent COD.

with or without sludge recycle. A recent study has also shown the validity of this model with both carbon and oxygen limiting conditions.29 In a well-mixed continuous culture system wit.h no sludge recycle, the rate of change in the density of microorganisms in the system is given by

dX

-=

dt

dS

Y-dt

k,X

and the empirical expression for the rate of waste (substrate) utilization, dS/dt, is

I n these equations, X is the concentration of microorganisms in the system; S is the substrate concentration in the system in terms of COD; Y is the yield coefficient or the maximum obtainable yield of cells based on COD utilized; k is the maximum specific substrate utilization rate; K , is the saturation constant based on COD; kd is the specific death rate or the endogenous metabolic rate constant for the cells. In this study the hydraulic retention time, 0 (0 = V / Q ) ,equals the solids retention time, 0, (0, = V X / Q X ) , where V = volume of

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SURUCU, ENGELBRECHT, AND CHIAN

reactor and Q = volumetric flow rate. Therefore, a mass balance for the microorganisms in the reactor system can be written as:

in which the net rate of change of microbial mass equals the net growth less washout. At steady state, d X / d t is equal t o zero and eq. (3) yields:

QV

=

y -ds- 1 dt X

- kd

(4)

or

where U is the “specific utilization rate,” or “food t o microorganism ratio)’ (see eq. (8) for details). Equation (4) shows that, in this reactor scheme, U and Bc are directly related to each other. By utilizing eqs. (1) and (4), the expressions for the effluent waste concentration (S) becomes;

Using eq. (4) and the expression of rate of utilization on a finite time basis, dS/dt can expressed as:

_ As At

5L (So- S ) V

where S o is the inflow substrate concentration based on COD and Q/V is essentially equal t o the reciprocal of Bc. The microorganism concentration, X , in the reactor can then be given by :

Equations (5) and (7) show that after the coefficients Y , kd, k , and K , have been defined for a given waste, microorganism population and temperature of operation, the effluent waste concentration, S , and microorganisms concentration, X , are direct functions of ec.

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RESULTS AND DISCUSSION Determination of Kinetic Coeficients To employ kinetic equations in the control of biological systems as shown in eqs. (5) and (7), the kinetic parameters ( Y ,k d , K,, and k ) must be known. Studies t o evaluate these kinetic coefficients within the mesophilic temperature range and under a variety of environmental conditions have shown that the values for Y , k d , and k , although different for many carbon sources, do not vary to a significant extent for most engineering purposes (Table II).28 The values listed for K , in Table I1 are higher for pure substrates than that for mixed substrates. The researchers who have studied the effect of temperature on the four individual parameters have pointed out that they are influenced significantly by t e m p e r a t ~ r e . ~ ~ ~ ~ ~ Therefore, it is necessary to determine these coefficients in the thermophilic temperature range if the kinetic equations are t o be used in controlling a thermophilic biological system. The kinetic parameters Y and Kd were determined by utilizing eq. (4),in which U , the specific substrate utilization rate, is equal to (So- S)/O,X. By substituting the latter intcreq. (4)for U so as to obtain the equation

a plot of i / e , versus U should give a straight line, intersecting the l / 6 , axis a t a point equal to k d , the coefficient of bacterial decay. The slope of the line will correspond to Y , the yield coefficient (Fig. 4). Similarly, k , the maximum specific substrate utilization rate, and K,, the saturation constant, can be obtained from the linearized form of eq. (2). By replacing dS/dt with (So- S)/O, and taking the reciprocal of eq. ( 2 ) and rearranging, the Lineweaver-Burk equation is obtained 1

e,.x (So- S )

-

K - ., - 1 k

s

1 +

i

(9)

A plot of O , - X / ( S o - S ) versus 1/S should give, again, a straight line intersecting the 1/S axis at a point equal to - l / K , and the O,.X/(S, - S) axis at the point corresponding to l / k (Fig. 5). From Figure 4,the yield Coefficient, Y , was calculated t o be 0.34 (mg of biomass/ mg COD utilized) and the decay coefficient, k d , was found to be 0.48 day-'. When compared with the yield values found by different researchers using glucose as the carbon source

0.048

0.045 0.18

0.087

0.67

0.48 0.65

0.42 0.59 0.67

aSee Lawrence and McCarty.2s

0.055

0.5

0.07

(day-')

Domestic Waste Domestic waste Skim milk Synthetic waste Glucose Glucose Domestic waste Synthetic

kd

Y (mg/mg)

Wastewater composition

Growth coefficients

>9.7

3.0 3.3 5.6

5.1

k (mg/mg day)

~.

Waste removal coefficients

22

355

100

K8 (mg/liter)

Kinetic Coefficients for Mixed Culture Aerobic Systems"

TABLE 11

COD

BOD5 BOD6 COD

BOD5 BOD6

BOD,

BOD6

Coeficient basis

10

20-21

Temperature ("C)

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1

-(S,-S) a. X

Fig. 4.

Determination of Y and kd.

(Table 11),the yield coefficient obtained in this study using thermophilic mixed culture is somewhat lower. However, this result is in agreement with the observations of Muck and grad^.^^ In their recent study on “temperature effects on microbial growth,” Muck and Grady30 found that the yield coefficient increased initially as

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t-

600

500

-

I 16' S Fig. 5 . Lineweaver-Burk plot.

the temperature of the culture was increased from loo to 20"C, but decreased with temperatures greater than 20°C. Therefore, from their observation, and from the higher endogenous metabolism rate at elevated temperatures, a lower yield coefficient would be expected with systems operated in the thermophilic temperature range than in systems operated under mesophilic conditions. Figure 4 shows that the decay coefficient, kd, under thermophilic conditions is about ten times greater than that associated with mesophilic conditions. This result is very much in agreement with the findings of Muck and Grady30 and Topiwala and S i n ~ l a i r . ~ ~ These authors have shown that kd is affected by the temperature in a manner which can be described by the Arrhenius equation. From the Lineweaver-Burk plot (Fig. 5 ), the saturation constant, K,, and the maximum sepcific substrate utilization rate, k , were found to be 740 mg COD/liter and 15.38 mg COD/mg-day, respectively. These values are comparatively higher than those reported for systems operated in the mesophilic temperature range (Table 11). A high K , value (740 mg COD/liter) indicates that the maximum rate of substrate utilization per unit mass of microorganisms will be reached

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at a much higher substrate concentration, i.e., 1480 mg COD/liter On the other hand, a high k value indicates that, in a unit time, more waste per unit quantity of biomass can be removed from the waste stream. Figure 6 compares the theoretical and experimental change in concentration of biomass as a function of SRT. The theoretical curve was obtained by utilizing eq. (7) for solids concentrations; the yield and decay coefficients were taken from Figure 4. Since the theoretical curve is in good agreement with the experimental data, it would appear that the kinetic model, which was developed for mesophilic microorganisms, is also applicable to the thermophiles. The agreement between the experimental and theoretical curves also shows that the kinetic coefficients determined for the system were valid.

SURUCU, ENGLEBRECHT, AND CHIAN

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Relationship Between Observed Cell Yield and SRT From eq. (7), it is apparent that there should be less biomass production with increasing SRT, 8,. Many investigators have reported a lower net cell production with longer SRT.28p32Figure 7 clearly shows that observed cell yield, i.e., net sludge production, decreases as the SRT increases. An observed cell yield of 0.28 (mg dry weight of cells/mg COD utilized) was observed with a cell residence time of 0.5 days. As cell residence time was increased to ten days, the cell yield decreased to a value of 0.06 (mg cells/ mg COD utilized). Factors which may contribute to the decrease in observed cell yield with increasing SRT are increased maintenance energy and cell death at longer SRT.

Effects of SRT on COD Removal Figure 8 shows the steady-state effluent soluble COD and percent COD removal for each experimental SRT. The percent COD removal was based on the total amount of organic matter in the influent medium. The data indicate that the maximum soluble organic matter removal, as determined by precent COD removal, was obtained for a SRT of five days or greater. It is seen from Figure 8 that 85y0 of the initial COD was removed with one day SRT, I

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S R T (days)

Fig. 7. Effect of SRT on net biomass production.

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Porcont COD Romoval 1003 !

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(Days)

Fig. 8. Steady-state COD values.

and COD removal reached 90% when SRT increased to two days. Finally, the removal of COD reached 95.5% for a SRT of five days.

Effect of SRT on Protein Content of Biomass In performing the continuous flow experiments, biomass was collected from each system operating at different SRT. The samples were collected when the systems were operating at steady state and were analyzed for total protein. The results of the effect of dilution rate, i.e., reciprocal of SRT, on the total protein content of the biomass are shown in Figure 9. It is interesting to note that the percent protein of the biomass decreased linearly with an increasing

SURUCU, ENGELBRECHT, AND CHIAN

1666

o 5

20

0 01

002

003

DILUTION

RATE

004

0 05

006

007

(hr’)

Fig. 9. Effect of dilution rate on percent protein content of biomass.

dilution rate. At the lowest dilution rate studied, e.g., 0.004 hr-1 (ten days SRT), the biomass contained approximately 45% protein. It decreased to 36% at a dilution rate of 0.057 hr-’ (0.75 day SRT). Herbert,33in his study on the “effect of growth rate on cell composition and morphology,” similarly observed that the percentage of total protein of the cells (Aerobacter aeTogenes) decreased with an increasing dilution rate. However, no explanation was given for this effect. This observation is significant if the biomass is going t o be used as a protein source, e.g., a supplement in animal feed. On the other hand, since apparent cell yield increased with an increasing dilution rate (Fig. 7), a trade-off of dilution rates against net protein yield can be made for achieving maximum production of protein. Figure 10 clearly shows that the amount of protein produced per unit COD removed, lb protein per lb COD removed, increases significantly with an increasing dilution rate. Efect of S R T on Oxygen Requirements

It is interesting to note from Figure 10 that the oxygen required per unit quantity of protein produced increases with a decreasing dilution rate, i.e., increasing SET. This occurred because, a t a higher dilution rate, only a relatively small fraction of the total amount of oxygen uptake was used for endogenous respiration; most of the oxygen was utilized for oxidation of substrate for energy which

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is needed for the synthesis of the biomass. However, a t lower dilution rates, auto oxidation of the biological solids becomes very important and increases the amount of oxygen consumed per unit quantity of protein produced. This observation also indicates that operation of the system a t high dilution rates is more advantageous for protein production. However, the opposite is true from the standpoint of wastewater treatment; in this case, a good quality effluent (Fig. 8) and minimum sludge production (Fig. 7) are desirable.

Economic Assessment

It is recognized that any viable industrial process depends greatly upon favorable process economics. The most critical factor associated with the recovery of SCP from wastewaters appears to be the separation costs, i.e., costs of dewatering, drying, etc. An attempt has been made t o give an economic analysis of the thermophilic process for SCP recovery based on information obtained from this study. It has been shown that a waste stream with a minimum of 7,500 mg/liter COD is required for the thermophilic (55-60°C) process to be self-sustaining with the heat produced by thermogenesis.l4 Therefore, the cost estimate was made on the basis of wastewaters containing 12,500 mg/liter and 25,000 mg/liter COD. This will provide a safety margin for the process to be self-sustained and demonstrate the effect of increased wastewater strength on the process economics. The effect of the size of the treatment facilities on the cost of SCP recovery was also determined. Table I11 shows the

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SURUCU, ENGELBRECHT, AND CHIAN TABLE 111

Cost Estimates of SCP liecovery from the Thermophilic Process of Waste Treatment Operating time (300 days/yr) Protein content (37% of solids) A. Wastewater flow rate, MGD B. COD of wastewaters, mg/liter C. Lb prot.ein produced/yr D. Production costs $/lb protein Aerobic treatment8 Thickening to 2% solids Membrane dewatering to 12% solids Drying to 9 5 9 9 % solids E.

0.1 1 25,000 12,500 25,000 12,500 5 X lo6 2.5 X lo5 5 X lo6 2..5 X lo6 3.4 1.9

3.6 2.2

3.0 1.1

3.2 1.3

5 .ii 8

7 8

4 6.8

6.8

Total costs of production Credit from waste disposal $/lbb protein

18.8

20.8

14.9

16.3

7.6

8.2

7.6

8.2

Net costs (D-E) $/lb protein

11.2

12.6

7.3

8.1

3

* SRT = 0.67 day, COD removal 80% (Fig. 8). b Surcharge rates applied: $104/million gal, $6.25/1000 lb COD, and $14.25/1000 lb suspended solids. Assume suspended solids were 5% of COD.

production cost of SCP in cents/lb of protein a t two different wastewater flow rates, i.e., 0.1 and 1.0 million gallons per day (MGD). The designed hydraulic detention time was 0.67 day, corresponding to a diltuion rate of 0.0625 hr-l. Since no cell recycle was anticipated, the SRT was equal to 0.67 day. From Figure 8 the estimated percentage removal of COD is 80 at a S R T of 0.67 day. Protein yield is estimated from Figure 10 t o be 0.1 lb protein per lb COD removed. Based on an operating time of 300 days per year, the total amount of protein produced per year is calculated for wastewaters having different concentrations a t different flow rates (Table 111). There are four major steps anticipated in the recovery of SCP from wastewaters; aeration, preconcentration, dewatering, and drying. The cost estimates given for each step include utility, chemicals, labor, maintenance, taxes, insurance, and depreciation. The latter was based on a 20 year straight line for stationary equipment, a ten year straight line for equipment with moving parts, and a three year straight line for membranes. The utility costs were based on 2 cents kWhr and $2/1000 lb steam. The interest payment on capi-

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tal investment is not included. Once the product value and sales expenses are established, the return on investment can be calculated and compared with the interest rate t o aid in decision making on the selection of the process. The total capital investment is estimated a t $0.25 and $1.0 million for the 0.1 and 1.0 MGD plants, respectively. The latter figure approximates t o the capital investment for the recovery system of a hydrocarbon SCP production plant of comparable size.Is Figure 10 was used to estimate power requirements for aeration. Seven pounds of oxygen are required per lb of protein produced a t a dilution rate of 0.0625 hr-' (Fig. 10). This corresponds t o a required oxygen transfer rate of 0.6 g/liter hr when fed with a wastewater having a COD of 25,000 mg/liter. The required power input is 1 hp/lOOO gal at an air flow rate of 0.2 w m and an oxygen utilization of 15yo. Approximately two-thirds of the costs involved in the aerobic treatment were for power. From Figure 7 the amount of biomass produced is estimated a t 0.25 lb/lb COD removed. This would result in a 0.5yocell suspension from a wastewater containing 25,000 mg/liter COD a t an 80% COD removal efficiency (Fig. 8). Because the concentration of the treated effluent is too low to be dewatered economically with membrane ultrafiltration and the tiny bacterial cells are too small to be immediately centrifuged, preconcentration is necessary prior to the dewatering step. Chemical precipitation with food gradc coagulants followed by clarification and thickening is visualized to bring the cell mass to an underflow concentration of 2%. An average loading rate of 20 lb/ft2/day on the thickener was used for the design.34 Preconcentration of bacterial cells with chemical precipitation and thickener is a common practice in pilot scale SCP productionP5 The selection of membrane ultrafiltration (UF) instead of centrifugation for the dewatering step was to demonstrate the feasibility of using the membrane process for SCP recovery. Membrane fouling is minimized a t a n operating temperature above 55°C where fluxes are increased ~ i g n i f i c a n t l y . ~An ~ average flux of 75 gal/ft2/day a t a feed flow rate of 30 gal/min through a 1 in. diameter tubular module was used for the design of the dewatering process. The power requirement was between 15-20 kWhr/1000 gal of water processed. Although a final cell concentration of 15y0 may be obtained with the U F process,36the design concentration was 12%. It is conceivable that centrifugation might be less costly, due to both lower equipment and operating costs as well as higher attainable cell concentration, e.g., up t o 20oJ, cell mass. The membrane process was chosen due

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to the availability of operating information. Also, it reprcsented a more conservative approach in estimating the cost for the entire SCP recovery step. Final drying will bc carried out using drum driers. The designed capacity of equipment was based on 2 lb dry solids/ft2/hr. The utility cost was based on 1.3 lb stcam per lb of water evaporated. It is anticipated that the use of a spray drier would cost somewhat morc. As shown in Table 111, more than 80% of the costs was associated with the solids recovrry steps. The total costs of SCP recovery was approximately 14-20 cents/lb protein depending upon the conccntration and volume of the wastewater. These figurcs are reasonable as compared with 23-30 cents per Ib protein produced from hydrocarbonsIs and 20 cents/lb protein from a waste stream of acid cheese whey containing an average COD of 60,000 mg/liter.37 However, this process becomes extremely promising if one considered thc credit received from paying a surcharge for the disposal of wastewaters into the municipal wastewater treatment plants. Using a typical surcharge rate of $104/million gallon of waste, $6.25/1000 lb COD, and $$14.25/1000 lb suspended solids as set by the Los Angeles County Sanitation the total credit is about 8 cents/lb protein. This results in a final cost of 7-12 cents/lb protein. This is almost one-half the cost of a lb of protein produced from conventional SCP plants. It is seen from Table I11 that thc costs associated with the SCl’ recovery are relatively more sensitive t o the size of the treatment plants than to the concentration of the wastewaters within the ranges studied. The slight differencts in separation costs of SCP between wastewaters having COD values of 12,500 and 25,000 mg/liter are mostly attributed to the preconcentration and dewatering strps. Little diffcrerice is anticipated with the final drying as steam consumption constitutes the bulk of the cost involved in this step. The difference becomes even smaller since the credit received on per lb protein basis is more with the wastewater having a louer COD concentration duc to thc built-in fixed charges on the disposal of the volume of ivastewater. It is apparent that a x-aste strength of approximately 12,500 mg/liter of biodegradable COD must be maintained for the process t o be self-sustaining a t the thermophilic temperature, and a flow rate of 0.1 JIGD for the process economics. However, the cost estimates made here are on the conservative side since the membrane process is used instead of the lo\\ er cost centrifugation process for dewatering.

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CONCLUSIONS Based on the findings of this investigation, the following conclusions may be drawn. The kinetic parameters K,, k , and kd for a thermophilic system were much higher than those of a mesophilic system while Y was somewhat lower. Over 90% removal of soluble COD was observed with a S R T of two days and i t improved with increasing SRT. The protein content of the biomass increases as the dilution rate decreases. However, because of a significant increase in the apparent cell yield with higher dilution rates, the net protein yield was found to increase with increasing dilution rates. A decrease in oxygen consumption per unit quantity of protein produced with increasing dilution rates also favors the economics of protein production. It was shown that the process was feasible for SCP recovery when the concentrations and flow rates of wastewaters were respectively above 12,500 mg/liter COD and 0.1 MGD while operated at a low SRT, e.g., 0.67 day. This was especially true for higher wastewater flow rates and concentrations and when credits for surcharges were taken into consideration. Nomenclature k maximum specific substrate utilization rate ((mg/mg)/day) kd specific death rate (day-') K , saturation constant (mg/liter) substrate flow rate (m3/day) S effluent substrate concentration (mg/liter) SO inflow substrate concentration (mg/liter) hydraulic retention time (day) 0 e, solid retention time (day), SRT U specific utilization rate ((mg/mg)/day) X cell concentration (mg/liter) V volume of reactor (m3) Y yield coefficient

Q

One of the authors (G. A. S.) was initially supported during the course of this work by a fellowship from the World Health Organization and later through a research appointment in the Dept. of Civil Engineering, University of Illinois a t Urbana-Champaign.

References 1 . R. I. Mateles and S. R. Tannenbaum, Eds., Singk-Cell Protein, The M I T

Press, Cambridge, Mass., 1968. 2. C. D. Callihan and C. E. Dunlap, U.S. Environmental Protection Agency Report SW-24C, 1971. 3. R. I. Mateles, J. N. Baruah, and S. R. Tannenbaum, Science, V , 1322 (1967). 4. W. D. Bellamy, Biotechnol. Bioeng., 16, 869 (1974).

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5. W. Hayes, The Genetics of Bacteria and Their Viruses, Blackwell Sciences Publishers, Oxford, 1964. 6. E. R. L. Gaughran, Bacteriol. Rev., 11, 189, 225 (1947). 7. J. T. Pfeffer, Biotechnol. Bioeng., 16,771 (1974). 8 . F. J. Stutzenberger, A. J. Kaufman, and R. P. Lossin Can J. Microbiol. 16, 553 (1970). 9. F. J. Stutzenberger, Appl. Microbiol., 22, 147 (1971). 10. D. L. Grawford, E. McCoy, J. M. Harkin, and P. Jones, Biotechnol. Bioeng., 15, 833 (1973). 11. G. J. Hajny, C. H. Gardner, and G. J. Ritter, Znd. Eng. Chem. 43, 1382 (1951). 12. E. Steel, Biochemical Engineering, The McMillan Co., London, 1958. 13. K. Kambhu and J. F. Andrews, J . WaterPolkct. Contr. Fed., 41, R127 (1969). 14. G. A. Surucu, Ph.D. Thesis, University of Illinois, Urbana-Champaign, Ill., 1975. 15. Perry, Chemical Engineers Handbook, 4th ed., McGraw Hill Co., New York, 1963, pp. 14-26. 16. R. Singleton, Jr. and R. E. Amelunxen, Bacteriol. Rev., 37, 320 (1973). 17. E. E. Harris, G. J. Hajny, and M. C. Johnson, Ind. Eng. Chem., 43, 1593 (1951). 18. A. E. Humphrey, Chem. Eng., 98 (1974). 19. Y. W. Han and V. R. Srinivasan, Part I, Final Report Under Res. Grant EC-00328, Louisiana State University Baton Rouge, LA 70803 (1970). 20. N. R. Smith, R. E. Gordon, and F. E. Clark, Agriculture Monograph, No. 16 U.S. Dept. of Agr. Nov. 1952. 21. Stanclard Methods for the Examination of Water and Waste Water, 13th ed. American Public Health Association, Inc., 1971. 22. 0. H. Lowry, J. Biol. Chem., 193, 265 (1951). 23. R. I. Mateles and E. S. K. Chian, Environ. Sci. Tech., 3, 6, 569 (1969). 24. J. Monod, Ann. Znst. Pasteur, 79, (1950). 25. A Novick and I. Szilard, Proc. Nat. Acad. Sci., 36, 708 (1950). 26. D. Herbert, Continuous Cultivation of Microorganisms, Proc. 2nd Symp., Prague, 1962, p. 23. 27, V. N. Uden, Ann. Rev. Microbiol., 23, 472 (1969). 28. A. W. Lawrence and P. C. McCarty, J . San. Eng. Div., ASCE, 96, SA3,757 (1970). 29. C. G. Sinclair and D. N. Ryder, Biotechnol. Bioeng. 17, 3, 375 (1975). 30. R. E. Muck and L. C. P. Grady, J . Env. Engr. Div., ASCE, 100,1147 (1974). 31. H. Topiwala and C. G. Sinclair, Biotechnol. Bioeng., 13, 6, 795 (1971). 32. J. H. Sherrard and E. D. Schroeder, Water Res., 6, 1039 (1972). 33. D. Herbert, Continuous Cultivation of Microorganisms, Proc. 1st Symp., Prague, 1958, p.45. 34. Black and Veatch Consulting Engineers, EPA Report 17090 DAN 10/71, 1971. 35. M. Rosenweig, Chem. Eng., Jan. 7, 2 (1974). 36. Abcor Incorporated, 341 Vassar St., Cambridge, Mass. 1975. 37. Milbrew, Inc. Junea, WI, Chem. Eng., March 17 (1975). 38. J. G. Kremer, Head, Industrial Waste Section, County Sanitation Districts of Los Angeles County, Whittier, Cal. 1975.

Accepted for Publication July 19, 1975