APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Mar. 1986, p. 498-503 Vol. 51. No. 30099-2240/86/030498-06$02.00/0Copyright CO 1986, American Society for Microbiology

Optimized Batch Fermentation of Cheese Whey-SupplementedFeedlot Waste Filtrate to Produce a Nitrogen-Rich Feed Supplement

for RuminantstM. D. ERDMAN'- AND C. ADINARAYANA REDDY2*

Departments ol 'Animatil Sciencel ainICl Microbiology ailidI Public Heajlthl,2 Mi(chliganl1l Statte UniversitY, Etast Lantisinig,Michigan 48824

Received 3 October 1985/Accepted 16 December 1985

An optimized batch fermentation process for the conversion of cattle feedlot waste filtrate, supplementedwith cheese whey, into a nitrogenous feed supplement for ruminants is described. Feedlot waste filtratesupplemented with cheese whey (5 g of whey per 100 ml) was fermented by the indigenous microbial flora inthe feedlot waste filtrate. Ammonium hydroxide was added to the fermentation not only to maintain a constantpH but also to produce ammonium salts of organic acids, which have been shown to be valuable as nitrogenousfeed supplements for ruminants. The utilization of substrate carbohydrate at pH 7.0 and 43°C was >94%within 8 h, and the crude protein (total N x 6.25) content of the product was 70 to 78% (dry weight basis).About 66 to 69% of the crude protein was in the form of ammonia nitrogen. Lactate and acetate were thepredominant acids during the first 6 to 8 h of fermentation, but after 24 h, appreciable levels of propionate andbutyrate were also present. The rate of fermentation and the crude protein content of the product were optimalat pH 7.0 and decreased at a lower pH. For example, fermentation did not go to completion even after 24 h atpH 4.5. Fermentation proceeded optimally at 43°C, less so at 37°C, and considerably more slowly at 23 and50°C. Concentrations of up to 15 g of cheese whey per 100 ml of feedlot waste filtrate were fermented efficiently.Fermentation of feedlot waste filtrate obtained from animals fed low silage-high grain, high silage-low grain,or dairy rations resulted in similar products in terms of total nitrogen and organic acid composition.

Cattle wastes account for about 70% of the >1.8 x 1012 kgof livestock wastes produced annually in the United States(2, 3, 10, 20, 34). Nearly 50% of these wastes are generatedin confined animal production systems such as cattle feedlotoperations (34). The disposal of large volumes of feedlotwaste represents a critical problem with respect to environ-mental pollution, a loss of large volumes of potentiallyutilizable nutrients (2 x 109 kg of total nitrogen), and afinancial drain to the livestock industry (4, 27). Feedlotwaste can be considered a valuable renewable resource ifproperly utilized. The recycling of feedlot waste as a live-stock feed ingredient would partially alleviate the disposalproblem and supplement our feed resources. A number ofreviews (1, 3, 10, 11, 18, 21) and reports regarding the use ofunfermented, ensiled (14, 25), or fermented (24, 29, 32, 33)feedlot waste as a livestock feed have been published.Ammoniated organic acid fermentation of agricultural

wastes into nitrogenous feedstuffs appears to be a novel andefficient solution to the problem of agroindustrial wastes(13). This approach has been successfully used for recyclingcheese whey as a crude protein supplement for ruminants (6,17, 28). This product has since been approved as a feedsupplement by the U.S. Food and Drug Administration(CFR 573-450). Reddy and Erdman (27) later described ananaerobic, ammoniated organic acid fermentation processfor the conversion of cattle feedlot waste filtrate into apotentially useful nitrogenous feedstuff for ruminants. In this

* Corresponding author.t Journal article no. 9097 of the Michigan Agricultural Experi-

ment Station.t Present address: U.S. Department of Agriculture, P.O. Box

748, Tifton, GA 31793.

process, feedlot waste filtrate, which contains 72% of thetotal nitrogen in feedlot waste but is deficient in a ferment-able energy source, was supplemented with either of twocomplementary carbohydrate-rich agricultural wastes:cheese whey (28) or starch recovered from potato-processing wastes (12). The combined substrate wasanaerobically fermented batchwise by indigenous microbiotain feedlot waste at 43°C and pH 5.5 for 24 h. The objectivewas to maximize the conversion of carbohydrate in thesubstrate into organic acids and to neutralize the organicacids with ammonia to produce their ammonium salts.Ammoniated organic acids were shown to be superior tourea and comparable to soybean meal as nitrogenous feedsupplements for ruminants (6. 13, 17). Advantages inherentin this approach, compared with processes for single-cellprotein production for wastes, were summarized previously(27).The purpose of this investigation was to study the effects

of pH, temperature, cheese whey concentration, and othervariables on the rate of fermentation and the types andquantities of organic acids produced during the fermentationand to define optimal operating conditions for the batchfermentation of feedlot waste filtrate.

MATERIALS AND METHODS

Substrate. Unless otherwise stated, fresh (.--1-day-old)feedlot waste, collected from finishing steers fed a high-silageration (88% corn silage, 12% soybean meal), was usedthroughout this study. The feedlot waste, which contained onthe average 16.5%, total solids, was diluted 1:1 (wt/wt) withwater, mechanically agitated for 30 min, and filtered througha screen (mesh size, 3 mm) to remove large particulate

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TABLE 1. Composition of unfermented and fermented feedlot waste filtrate before and after supplementation with cheese whey pow,der

Feedlot walste c:ondition" Carbohyvdrate Total N Ammoniai N Total Organic acids (,tmol/ml)(g/ltX) ml) (g1100 ml) (g/t(X) ml) solids ('7) Lactic Acetic Propionic

Unfermented (0.11 0.28 0.02 5.5 2.85 21.95 3.80Plus cheese whey powder 3.94 0.33 0.07 6.6 16.30 21.13 5.80

Fermented 0.12 0.28 0).02 5.4 1.10 11.95 2.30Plus cheese whey powder 0.21 ().88 0.59 6.3 333.70 129.53 27.10

Cattle feedlot waste filtraite was termented voth or without 5 g of cheese \vhey powder per 1t)() ml at 43WC and pH 7.)) fo- 8 h.

material. The resulting feedlot waste filtrate, which contained7 to 9% total solids, was supplemented with cheese wheypowder, fresh acid whey, or deproteinized whey (MichiganMilk Producers, Ovid, Mich.). In most experiments, cheesewhey powder was used as the substrate because of itsconvenience and uniformity. Feedlot waste filtrate wasroutinely supplemented at the rate of 5 g of cheese wheypowder per 100 ml unless otherwise indicated. Supplemen-tation with cheese whey powder at this level contributed 0.05g of total nitrogen and 3.83 g of lactose per 100 ml of feedlotwaste filtrate.

Fermentation procedures. Laboratory-scale. nonaseptic,anaerobic, 20-liter batch fermentations were performed aspreviously described (9, 27), except that the fermentationswere conducted at pH 7.0 and 43 + 0.5°C unless otherwiseindicated. The pH in the fermentor was maintained at apreset level, as described previously (27), with an automaticpH controller assembly connected to an electric solenoidvalve. As the pH drops, the solenoid valve opens and allowsthe addition of ammonium hydroxide. Once the pH rises tothe preset level, the controller terminates the electricaltransmission to the solenoid valve, and the addition of basestops. Indigenous microbiota in feedlot waste filtrate servedas the inoculum.

Analytical procedures. Samples (80 ml) were collected inglass screw-cap bottles at various times during the fermen-tation and stored at -18°C until analyzed. All samples wereanalyzed for total nitrogen, ammonia nitrogen, lactose, totalsolids, and volatile and nonvolatile acids. The total-nitrogenconcentrations of whole samples were determined by themicro-Kjeldahl procedure (31). The total solids were deter-mined gravimetrically by the drying of 50- to 70-g samples at50°C and reduced pressure to a constant weight. The super-natant obtained by centrifugation (20,000 x g for 30 min at4°C) of the samples was used to determine concentrations ofvolatile and nonvolatile fatty acids in a gas chromatographequipped with a flame ionization detector and temperatureprogramming (Model 5730A; Hewlett-Packard Co., PaloAlto, Calif.). Volatile acid concentrations were determinedwith a column packing of 3% Carbowax 20M (Union CarbideCorp., New York, N.Y.)-0.5% H3PO4 on 60/80 Carbopack B(Supelco, Inc., Bellefonte, Pa.) by the procedure of DiCorciaand Samperi (7), except that the column temperature. ini-tially 100°C, was raised (4°C/min) to 200°C. Nonvolatileacids were esterified and extracted by the procedure ofHoldeman and Moore (16) and were injected onto a columnof 10% SP-1000-1% H3PO4 on 100-120 Chromosorb W AW(Supelco). The column temperature, initially 90'C. wasraised (4°C/min) to 130°C. The determination of lactoseconcentration by the procedure of Dubois et al. (8). asmodified by Montgomery (23), and the determination ofammonia as described by Oser (26) were performed on 5.0ml of supernatant which had been treated with 1.0 ml of 1.96M 5-sulfosalicylic acid, incubated at ambient temperature for

30 min, diluted (1:1). and clarified by centrifugation at 20.000x g for 30 min. The samples were periodically checked forthe presence of pathogens at the Veterinary Clinical Diag-nostic Laboratory of Michigan State University.

RESULTS

Rate study. The rate of decrease in lactose concentrationand the rates of increase in ammonia nitrogen and totalnitrogen concentrations during the fermentation at 43°Cindicated that most of the whey lactose (>94%) was metab-olized within 6 h. A decrease in the concentration of lactoseresulted in a concomitant increase in the concentration oforganic acids, ammonia nitrogen, and total nitrogen (Table 1;Fig. 1). Organic acids accounted for >95% of the lactoseutilized. Ammonia nitrogen content and total nitrogen con-tent increased 2.6-fold during the fermentation, and theproduct contained 70 to 78% crude protein on a dry weightbasis. Ammonia nitrogen accounted for 66 to 69% of the totalnitrogen. The data demonstrated that the progress of thefermentation during the first 6 to 8 h could be monitoredefficiently by a measurement of either a decrease in lactoseconcentration or an increase in ammonia nitrogen or totalnitrogen concentrations. In control fermentations in whichfeedlot waste filtrate was not supplemented with cheesewhey powder, there was no significant increase in totalnitrogen or ammonia nitrogen concentrations, and there wasonly a minimal increase in organic acid content.

Effect of pH. The effect of pH upon fermentation. asmeasured by the rate of increase in total nitrogen is pre-sented in Fig. 2A. Fermentations maintained at pH 6.5, 7.5(data not shown), or 7.0 had the fastest rate of total-nitrogenincrease and lactose utilization (Fig. 2B) and were essen-

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FIG. 1. Rate of lactose utilization ( 0 ) and rates of increase inammonia nitrogen (E) and total-nitrogen (0) concentrations duringfermentation of feedlot waste filtrate supplemented with 5.0 g ofcheese whey powder per 100 ml at 43°C and pH 7.0.

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500 ERDMAN AND REDDY

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FIG. 2. Effect of pH 4.5 (0), 5.0 (E), 5.5(0), 6.0 (A), and 7.0(0 ) on the rate of increase in total-nitrogen (A) and lactoseutilization (B) during the fermentation of feedlot waste filtratesupplemented with S g of cheese whey powder per 100 ml at 43°C.

tially complete within 6 to 8 h. Fermentations maintained atpH 4.5 and 5.0 had the slowest rate of total-nitrogen increaseand lactose utilization. The fermentation maintained at pH7.0 was considered optimal, however, because largeramounts of total nitrogen were present in this product thanthat obtained at pH 6.5 (data not shown), and nitrogen lossesduring storage were lower in the product formed at pH 7.0than were present in the product formed at pH 7.5 (data notshown).

Organic acid composition was affected rather dramaticallyby pH and duration of fermentation (Fig. 3A to C). At pH 7.0(Fig. 3C), lactate and acetate were the predominant acidsafter 6 to 8 h and accounted for 72 and 26%, respectively, ofthe total acids produced. On the other hand, lactate, acetate,propionate, and butyrate were predominant after 24 h (37,23, 3, and 37%, respectively). Somewhat similar results wereobtained with fermentations conducted at pH 5.5 (Fig. 3B),6.0, and 7.5 (data not shown). In contrast, the fermentationswere essentially of the homolactic type (5) at pH 4.5 (Fig.3A) and 5.0 (data not shown).

Effect of temperature. Optimum fermentation, as deter-mined by the most rapid increase in total nitrogen and thehighest total-nitrogen concentration after 5 h, occurred at43°C, and the fermentation was essentially complete within 6h, whereas fermentations maintained at 37°C required 8 to 10h for completion (Fig. 4). Fermentations conducted at 23 and50°C were incomplete even after 20 h. When fermentationwas initiated at ambient temperature (20 to 23°C) and thefermentation temperature was not controlled, there was aninitial lag period of about 8 h followed by rapid fermentationfor between 10 and 20 h. In this fermentation, the tempera-ture rose from 23°C at zero time to 33°C at the peak offermentation (18 to 20 h after initiation), indicating that aconsiderable amount of heat was being generated endoge-nously. Acetate and lactate accounted for 38 and 58%,respectively, of the total acids after 24 h of this fermentation.The pattern of acid production during the first 6 to 8 h infermentations conducted at 37°C was comparable to thepattern seen in fermentations conducted at 43°C. However,after 24 h, lactate, acetate, propionate, and butyrate ac-counted for 67.8, 16.0, 3.7, and 10.1%, respectively, of thetotal acids at 37°C. In contrast, at 43°C, lactate, acetate,propionate, and butyrate accounted for 36.2, 37.3, 11.8, and13.2%, respectively, of the total acids after 24 h of fermen-

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FIG. 3. Effect of pH on concentrations of organic acids during fermentation of cheese-whey-powder-supplemented feedlot waste filtratepH: 4.5 (A), 5.5 (B), and 7.0 (C). Other conditions were as described in the legend to Fig. 1. Symbols for acids: acetic (0), propionic (O),butyric (O), and lactic (A).

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FIG. 4. Effect of temperature on the rate of increase in totalnitrogen during the fermentation of feedlot waste filtrate supple-mented with 5 g of cheese whey powder per 100 ml and maintainedat pH 7.0. Symbols: temperature not controlled (0). 23'C (@). 37TC(E). 43°C (b), and 50°C (A).

tation at 43°C (Fig. 3C). In fermentations conducted at 23and 50°C, the total-organic-acid production was consider-ably lower than that of fermentations conducted at 43°C (Fig.4). Lactate and acetate accounted for 75.7 and 19.3% of thetotal acids, respectively, after 24 h at 23°C, whereas in

fermentations maintained at 50°C, acetate and butyrateaccounted for 66.6 and 31.1% of the total acids, respectively.

Effect of initial concentrations of cheese whey powder.Fermentation with initial cheese whey powder concentra-tions of 5 or 10% proceeded rapidly and went to completionin 8 h. With an initial cheese whey powder concentration of15%, 16 h was required for completion of the fermentation,whereas with an initial cheese whey powder concentration of20%, the fermentation was incomplete even after 32 h (datanot shown). Similar to the fermentations supplemented with5% cheese whey powder, lactate and acetate were thepredominant acids (78.8 and 18.6%, respectively) in fermen-tations supplemented with 10% cheese whey powder. Simi-lar results were obtained in fermentations supplementedwith 15% or 20% cheese whey powder (data not shown). Infermentations initially supplemented with 0, 5, 10. 15, and 20g of cheese whey powder per 100 ml, the total organic acidsconcentrations at 24 h (in micromoles per ml) were 40, 501,1,000, 1,314, and 1,040, respectively. Although we routinelysupplemented feedlot waste filtrate with cheese whey pow-der because it is a uniform and convenient substrate, we alsoconducted several fermentations in which fresh whey or

deproteinized whey was used as the supplemental carbohy-drate source. These were similar to fermentations supple-mented with initial cheese whey powder concentrations of5% in all parameters tested (data not shown).

Effect of ration composition. The objective of the rationcomposition experiment was to determine the variation, ifany, in fermentation rate and product composition whenfeedlot waste filtrate from animals fed low silage-high grain,high silage-low grain, or typical dairy rations was used. Noappreciable differences were observed (data not shown) inthe rates of fermentation, as determined by the rate ofincrease of total nitrogen, when feedlot waste filtrate from

animals fed one of the three different rations was fermented.The rates of production and the composition of organic acidswere similar for all three fermentations.

Effect of manure storage. In the manure storage experi-ment, fermentation of feedlot waste filtrate derived fromfresh manure and that of feedlot waste filtrate derived frommanure stored for 91 days outside at ambient temperature inlate Fall were compared. Both fermentations were supple-mented with 5 g of cheese whey powder per 100 ml offermentation broth. The products obtained in both caseswere similar in total nitrogen and ammonia nitrogen content.except that 20 h was required for complete fermentation offeedlot waste filtrate from stored manure as compared with 8h for complete fermentation of feedlot waste filtrate fromfresh manure. When feedlot waste filtrate derived fromstored manure was supplemented with 10% fresh feedlotwaste filtrate, the fermentation duration and the productobtained were similar to those observed with feedlot wastefiltrate derived from fresh manure (data not shown).

Pathogen detection. Periodic tests for the presence ofaerobic and anaerobic pathogens after fermentation wereperformed throughout these studies. No aerobic oranaerobic pathogens were detected.

DISCUSSIONThe results showed that ammoniated organic acid fermen-

tation of feedlot waste filtrate initially supplemented with 5or 10% cheese whey powder could be efficiently carried outbatchwise at pH 7.0 and 43°C with indigenous microbial floraas the inoculum. Under these conditions, fermentation of theadded substrate was essentially complete in 6 to 8 h. Thisoptimized batch process is about three- to fourfold morerapid than the original process (27). The process describedhere is also 6 to 12 times faster, and the crude proteincontent of the product is much higher than that obtainedduring previously described aerobic fermentation of feedlotwaste filtrate by fungi and streptomycetes (33), Triclhoderina*'iride (15. 19, 24), or indigenous microbiota in feedlot waste(29, 32).

Feedlot waste filtrate is known to contain 70 to 80% of thetotal nitrogen originally present in feedlot waste (30). Thus,the process described here allows the recycling of approxi-mately 20% of the total solids and most of the total nitrogenoriginally present in feedlot waste. Simultaneously, theprocess also allows the recycling of a complementary wastecarbohydrate source such as cheese whey, which also con-stitutes a serious disposal problem for the dairy industry(28). Cheese whey primarily contributes lactose to the fer-mentation, while feedlot waste filtrate, which is deficient incarbohydrate, primarily provides inoculum and growth fac-tors. Although we used cheese whey powder as a convenientcarbohydrate source in this study, fresh, acid cheese wheyor deproteinized whey were comparable to cheese wheypowder as supplemental carbohydrate sources. Further-more, previous studies showed that cheese whey could bereplaced by other carbohydrate sources such as potatostarch recovered from potato-processing wastes (12), molas-ses, and corn starch (27).The fermentation pH greatly affected the production rate

and relative concentration of different organic acids; thissuggested dynamic changes in microbial populations andproduct quality during the fermentation. For example, at pH4.5 and 5.0, the fermentations were essentially homolactic,whereas at pH 5.5, 6.0, 6.5, 7.0, and 7.5, lactate and acetatewere the predominant acids after 6 to 8 h of fermentation;thereafter, lactate appeared to undergo secondary metabo-

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502 ERDMAN AND REDDY

lism as demonstrated by the rather dramatic decrease in

lactate concentration and the increases in the concentrationof propionate and butyrate. Apparently, cocultures ofacidogens and methanogens are active during this period (8to 24 h), because in addition to shifts in concentrations ofacids, we also observed (qualitatively) methane production.The total-nitrogen concentration reached the maximal levelwithin 8 h of fermentation at 43°C and pH 7.0. Therefore, itis desirable to terminate the fermentation at 6 to 8 h, whenlactate concentration is at its peak. Termination of thefermentation before further metabolism of lactate to otherproducts also permits conservation of most of the substratecarbon in the product.

Fermentation at the optimum temperature of 43°C wouldallow maximum recycling of feedlot waste in the minimumtime. This may be important at sites where the magnitude offeedlot waste accumulation is great. However, if largefermentor capacity or relatively limited amounts of feedlotwaste were available (all other things being equal), it mightbe more desirable to ferment feedlot waste filtrate at ambienttemperature (20 to 23°C), without any temperature control,to save energy and production costs.The optimized batch fermentation process described here

for the production of ammoniated organic acids is an attrac-tive alternative to the disposal of two important agroindus-trial wastes, cattle feedlot wastes and cheese whey. It issimple, efficient, and easily adaptable to the processing offeedlot waste at the source. The process is anaerobic, thuseliminating high equipment costs involved in aeration. Mostof the substrate carbon is conserved as fatty acids, which are

good sources of energy for the ruminant. There is no need tosterilize the medium or to supplement it with growth factors,and separate inoculum need not be added; thus, furtherreduction in operating costs are achieved.The potential for animal and human health problems

related to the recycling of animal wastes by feeding has beendiscussed previously (11, 22). In this study, no aerobic or

anaerobic pathogens were detected. These and other results(32, 33) suggest that feeding fermented feedlot waste to cattledoes not pose an unacceptable risk to animal or humanhealth. However, more-detailed investigations are needed toconclusively establish the safety and efficacy of this product.

ACKNOWLEDGMENTS

We thank Amanda Meitz for her technical assistance.This research was supported in part by a grant from the Michigan

Agricultural Experiment Station and by grant 616-15-61 from theU.S. Department of Agriculture.

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2. Azevedo, J., and P. R. Stout. 1974. Farm animal manures: an

overview of their role in the agricultural environment. CaliforniaAgriculture Experimental Station Extension Service Manual no.

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3. Bhattacharya, A. N., and J. C. Taylor. 1975. Recycling animalwastes as a feedstuff: a review. J. Anim. Sci. 41:1438-1457.

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30. Sloneker, J. H., R. W. Jones, H. L. Griffin, K. Eskins, B. L.Bucher, and G. E. Inglett. 1973. Processing animal wastes for

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34. Yeck, R. G., L. W. Smith, and C. C. Calvert. 1975. Recovery ofnutrients from animal wastes-an overview of existing optionsand potentials for use in feed, p. 192-194, 196. In Managinglivestock wastes. Proceedings of the 3rd International Sympo-sium on Livestock Wastes. American Society of AgriculturalEngineers, Urbana-Champaign, Ill.

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