Ruminal waste stream as a source of industrial chemicals

Resources, Conservation and Recycling 41 (2004) 215–226

Ruminal waste stream as a source of industrial chemicals Ajoy Koppolu∗ , L. Davis Clements1 Biological Systems Engineering, LW Chase Hall, University of Nebraska-Lincoln, Lincoln, NE 68583, USA Received 8 January 2003; received in revised form 3 October 2003; accepted 31 October 2003

Abstract Acetic, propionic, and butyric acids, which are volatile fatty acids (VFAs), are present in significant concentration in the rumen of cattle and other ruminant animals. The chemical oxygen demand (COD) burden carried by rumen wastes and ways to salvage the value-added enzymes, ␣-amylase and cellulases, which are present in substantial quantities are presented. An average COD for grain/grass-fed animals was 50 g/l and for cobs/alfalfa-fed was 23 g/l. The effect of four diets (grain, grass, cobs, and alfalfa) on VFAs concentration and on the COD for rumen fluid is reported. Enzyme concentrations, protein content (0.22–1.15 mg/ml), and ionic strength (86.4–113.8 mM/l) of rumen fluid are also mentioned. The activity of cellulase (31–40 international unit per liter) (IU/l) was not very different for any diet, but grain-fed animals showed substantially greater amounts of ␣-amylase (500 IU/l). The average pH for all the diets was about 6.5. Grain and grass-feds produced more protein than cobs and alfalfa-fed animals. Product separation and recovery of these chemicals as co-products from ruminal fluid can be an attractive alternative, which not only reduces the COD processing costs (US$ 2,000,000 per year), but can also bring profits from the saleable recovered products (a total of US$ 1,114,143 per year, i.e., US$ 698,610 from calcium magnesium paunchate (CMP), US$ 142,533 from ␣-amylase, and US$ 273,000 from cellulase). A review of recovery processes applicable to volatile fatty acids and enzymes is also included. © 2003 Elsevier B.V. All rights reserved. Keywords: Volatile fatty acids (VFAs); ␣-Amylase; Cellulase; Chemical oxygen demand (COD); Ruminal fluid; Economics

∗ Corresponding author. Present address: Technical Services, Novartis Consumer Health, Inc., 10401 Highway 6, Lincoln, NE 68517, USA. Tel.: +1-402-467-8939; fax: +1-402-467-8833. E-mail address: [email protected] (A. Koppolu). 1 Present address: Renewable Products Development Laboratory, 5100 N 57th, Lincoln, NE 68507, USA.

0921-3449/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.resconrec.2003.10.002

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1. Introduction Approximately 32 million cattle are slaughtered annually in the US for beef consumption. Each animal contains between 100 and 150 l of complex solid–liquid slurry in its rumen–reticulum chamber (Czerkawski, 1986). The gastrointestinal tract of these “ruminant” animals has a large organ, the rumen–reticulum chamber, inhabited by a complex microbial population, whose function is to degrade fibrous food before it reaches the true stomach. The rumen contents have a high biological oxygen demand/chemical oxygen demand (BOD/COD) and are considered a manure, called “paunch manure.” The total rumen resource is about 4 billion liter of fluid annually. This material presently constitutes a serious waste management problem for the beef processing industry. The industry must dispose of the rumen contents, incurring an unavoidable operating cost to the meat processor. Some of the contents of the rumen include undigested food, bacteria, fungi, volatile fatty acids (VFAs), enzymes, and water. VFAs in the rumen fluid consist primarily of acetic, propionic and butyric acids (APBs). Hungate (1966) indicated an average VFA concentration of 100 mM/l in cattle, with approximately 99% of this being APBs. The nature of the feed seems to affect the relative proportions of VFAs, suggesting a nutritional role of volatile fatty acids (Bergman, 1990). Dijkstra (1994) studied VFAs absorption from the rumen, and the possibilities of predicting the type of VFAs formed in the rumen. Mackie et al. (1984) worked with sheep rumen and showed the variation of VFAs concentration with time after feeding. Fiber (mainly cellulose and hemi-cellulose) is an important component of the ruminant diet. The microorganisms that break down this substrate are termed “cellulolytic.” The microorganisms exist symbiotically and act synergistically in metabolizing the food to sugars first and later to acids and alcohols. These rumen microbes secrete enzymes into the medium (rumen–reticulum chamber). Some of the enzymes, like amylases and cellulases, play an important role in depolymerizing the feed diet and are produced by ruminal bacteria such as Streptococcus bovis (Cotta, 1992) and by fungal species, among which are Neocallimastix frontalis, Neocallimastix patriciarum, Piromonas communis, etc. (Mountfort, 1987). Of the extra-cellular enzymes detected, the ones acting on cellulose have been studied in detail (Teunissen et al., 1993; Wood et al., 1986; Wood et al., 1995). Before applying any separation method, the waste stream should be characterized for its biological oxygen demand, ionic strength, and the concentration of various solutes present in it. BOD or chemical oxygen demand would indicate the strength of the waste stream and determination of the concentration of different solutes and ionic species would help in identifying the recovery needs and hence the selection of various separation methods. In this study, the ruminal fluid is characterized for its COD strength, VFAs load, total protein content, and concentration of enzymes such as ␣-amylase and cellulase. Four diets (grain, grass, cobs, and alfalfa) were fed to fistulated animals and the effect of diet and time after feeding on VFAs concentration and on the COD for rumen fluid was determined. COD was measured using a HACH COD reactor. Rumen fluid samples were analyzed for VFA content using capillary gas chromatography. ␣-Amylase activity was measured using p-nitrophenyl maltoheptaoside, while cellulase activity was measured using the filter paper method. Total protein content was measured using bovine serum albumin as the standard.

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A new approach to determine the percentage of COD contributed by the VFAs in the filtered supernatant to the total COD of the rumen fluid is presented. This approach facilitates using COD data as an index for correlating VFA quantities in rumen, as well as an indicator of demand for oxygen in the effluent (rumen) treatment system. Useful data can be obtained using this approach for designing a separation process. Presently, liquid waste streams from animal processing industries are treated in wastewater treatment plants. Recently, Westerhout et al. (1998) explained the economic and technical importance of utilizing waste and product separation from wastes. Before process development for recovering useful products from ruminal waste stream is considered, assessment of the BOD burden and its treatment costs is essential. If the treatment costs are off-set by the product development costs to a large extent, then product recovery from rumen contents becomes an attractive solution. Analysis of the attractiveness of product recovery from the rumen contents is presented in this paper.

2. Materials and methods Many methods have been used in the identification and separation of different products of anaerobic fermentation and rumen fluids. HPLC, gas-liquid and gas chromatography (GC) have been used for quantification of fermentation products (Brotz and Schaefer, 1987; Ehrlich et al., 1981; Playne, 1985). Teunissen et al. (1993) used an improved method of chromatography for simultaneous determination of many components of biological samples. Various materials and methods used in this study are presented below. 2.1. Sample preparation Rumen samples were collected from fistulated cows at different time intervals. Samples were filtered using cheesecloth to separate the undigested matter. The filtrate was centrifuged at 3000 × g for 30 min to separate the cell mass (wet pellet) and the supernatant. The supernatant thus obtained was filtered using Whatman filter #4 under suction to remove any micro particles. Samples were thus clear solutions without solid matter. Fig. 1 shows the preliminary clarification steps for the rumen samples before they were used for analysis. Different diets produce VFAs and enzymes in different amounts in the rumen fluid. Rumen samples obtained for the rations grain, grass, corncobs and alfalfa were analyzed. Each rumen sample was analyzed in duplicate after subjecting the sample to the sample preparation steps mentioned above. 2.2. Standard solutions Known quantities of various volatile fatty acids of analytical grade were used. Individual acids (APBs) were used in different molar concentrations for GC and COD analysis. These acids were mixed in a fixed proportion (A:P:B = 60:25:15) by weight to determine stoichiometric COD values, as well as for injections in the GC. The quantities in which they were mixed mimicked the proportion of acids that were found in filtered supernatant liquor and the literature (Hungate, 1966).

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Fig. 1. Mass balance for rumen samples. The supernatant and pellet streams subjected to COD, GC, protein, enzyme, and AAS analyses are shown along with their volume percentages. The samples were centrifuged at 3000 × g for 30 min after a preliminary filtration step.

2.3. Gas chromatography A HP 6890 GC, equipped with a flame ionization detector (FID), capillary inlet system, and GC protector was used for sample analysis. The chromatograph was controlled and interfaced to a HP Vectra PC and the HP 3365 ChemStation quantitative analysis and report making software. The separation was carried out with a 0.25 mm i.d. × 30 m × 0.25 ␮m HP-Innowax capillary column bonded with a cross-linked polyethylene glycol. Sample solution (ca. 1.0 ␮l) was loaded into the injection port at 280 ◦ C with a split ratio of 1:40 using electronic pressure control. The initial oven temperature of 120 ◦ C was held for 1 min and then programmed to increase to 265 ◦ C at 10 ◦ C/min. The column was thus held for 2 min at the final temperature. The eluent was detected with an FID at 300 ◦ C. Helium was used as the carrier gas. The inlet pressure of the carrier gas was controlled at 163 kPa and the linear velocity was 42 cm/s at the initial column-oven temperature. Calibration curves were constructed to measure the composition of rumen samples with respect to VFAs. Weight percent of the mixture and the relative amounts of individual acids were also varied to estimate the composition and acid strength of the samples. Rumen samples for gas chromatography were filtered through a 0.25 ␮m membrane before injections. 2.4. COD, ionic strength, and charged species measurement HACH’s USEPA approved COD apparatus was used to determine the chemical oxygen demand. Each COD vial contained 3 ml of prepared reagent, which follows APHA (1980) standard methods. A chloride inhibitor and a catalyst to promote reactions of organic compounds, which resist digestion, were present in the prepared COD reagent. A volume of 0.2 ml of the sample, from either the filtered supernatant or pellet, was added to the vial. The COD reactor was maintained at the digestion temperature of 150 ± 2 ◦ C and the reaction was carried out for 2 h. After digestion, the vials were analyzed with HACH’s DR100 colorimeter to read the results in milligram per liter. Conductivity of supernatants for

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various feeds collected after 2 h of feeding was measured using a conductivity meter. Ionic strength was calculated using conductivity values (Sparks, 1995). Concentrations of metal were measured using atomic absoprtion spectroscopy, and ionic species were measured using ion selective electrodes. 2.5. Enzyme and protein assays The ␣-amylase activity was assayed using p-nitrophenyl maltoheptaoside as substrate (Rauscher et al., 1985) with p-nitrophenol as the reaction product. The cellulase activity was determined by the filter paper assay method, which gives reducing sugar as the final product (Ghose, 1987). The final activities are expressed in concentration units as international unit per liter. One international unit of amylase and cellulase is defined as the amount of enzyme required to release 1 ␮mol of product per minute. Protein content for the four different diets was quantified by the method of Lowry et al. (1951) using bovine serum albumin as the standard.

3. Results and discussion 3.1. COD, ionic strength, and VFAs load The mass balances for the various streams that were subjected to the processing step are detailed in Fig. 1. The retentate (30–40% by volume) after the first step is mostly comprised of undigested wet solids. This material can be re-used as feed for the animals after drying or it can be burned or gasified as a waste management/energy production strategy. The two streams exiting from the centrifugation step of the clarification process contain effluents with a high COD value and valuable chemicals. The filtrate stream is 88–95% (by volume) rumen fluid, consisting of water, most of the protein, and VFAs, and the remaining 5–12% (wet pellet) is made up of cell mass, some protein, water, etc. The cell mass and microbial consortium which forms the pellet stream can be utilized for many purposes (Demain and Solomon, 1986). The average composition and the weights of individual acids present in the rumen VFA are summarized in Table 1. For all the diets, higher acids (like valeric, isovaleric) account for less than 5% of the total weight of acids. Acetic acid is found in greater quantities compared to the other acids. It was observed that grass and alfalfa roughage diets, because of their component composition, produce more acetic acid than concentrate diets like grain Table 1 Average composition of VFAs from rumen samples Acids

Composition (%)

Weight (g/l)

Acetic Propionic Butyric Higher

55–62 15–25 10–15 2–5

3 1.25 0.5 0.25

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Table 2 Chemical oxygen demand for four diets taken 2 h after the animals were fed Diet

Grain Grass Cobs Alfalfa

Rumen fluid

Filtered supernatant (vol%)

Wet pellet

COD (g/l)

Volume (l)

COD (g/l)

Volume (l)

COD (g/l)

Volume (l)

51 24 50 22

100 100 100 100

25 15 23 13

88 91 90 93

240 110 294 140

12 9 10 7

The majority of the COD burden resides in pellets which contain most of the cell mass, some protein, and other organics. Filtered supernatant consists mainly of volatile fatty acids, proteins, and water. The COD values depend on the type of feed and its composition. From the values the ruminal fluid stream can be termed as “strong” sewage (Metcalf and Eddy, 1972).

or cobs, hence, less propionic and butyric acids. Nevertheless, the values in Table 1 represent the average composition for all four diets. These numbers are in agreement with literature (Barnett and Reid, 1961). COD of the rumen fluid, filtered supernatant and wet pellet was measured using a HACH 45600 COD reactor. The measured values are shown in Table 2 for the diets studied in this work. The COD of rumen fluid for grain and cobs diet (which is similar to actual feedlot diet) is about 50 g/l. This means that a single animal contributes more than 5 kg of COD consisting of most of the cell mass, some protein, and other organic molecules. The COD of the filtered supernatant varies between 13 and 25 g/l, depending on the diet. Filtered supernatant from the grass and alfalfa diets has a lower COD when compared to the grain and cob diets. The pellet carries 110–294 g/l of the COD burden. Pellets from grain and cob diets have a higher COD when compared to grass and alfalfa diets. For grain and cobs diets, the wet pellet carries 57% of the rumen fluid COD and for grass and alfalfa diet 44% of rumen fluid COD is present in the wet pellet. Model solutions of acids were used to construct calibration curves to estimate the contribution of COD from VFAs towards the measured values of COD of the filtered supernatant. Individual acids were used in different concentrations (mM/l) and their COD was measured. The data collected using acetic, propionic, and butyric acids is shown in Fig. 2. Acetic, propionic, and butyric acids were mixed in the ratio of 60:25:15 by weight to form model solution mixtures ranging in concentration from 40 to 120 mM/l. CODs for these mixtures were measured and are plotted in Fig. 2. The fixed acid ratio was selected based on the APBs concentration in actual filtered supernatant rumen samples and the literature (Hungate, 1966). The average measured concentration of APBs in the filtered supernatant samples used was 71.5 mM/l and the average COD was 19,000 mg/l. The COD of the acid mixture in the model solution corresponding to 71.5 mM/l is read from Fig. 2 and is equal to 6750 mg/l. The difference in the COD between the filtered supernatant sample and the model solution containing APBs is attributed to substances other than APBs. It can be concluded that 36% of the COD in the filtered supernatant is a result of the presence of APBs, while the remaining 64% is from other substances. The theoretical COD of the given model solution mixture of APBs is 6445 mg/l.

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20000 acetic acid (100%) propionic acid (100%) butyric acid (100%) mixture (measured)

17500

COD (mg/L)

15000 12500 10000 7500 5000 2500 0 0

40

80 Acid concentration (mM/L)

120

160

Fig. 2. Calibration curves for COD determination. All samples were digested at 150 ◦ C for 2 h. Individual components were calibrated first for their COD content and later a mixture of known composition was evaluated.

A similar analysis can be done using the individual acid curves in Fig. 2. The measured COD values for individual acids (model solutions) from the graph are 2800 mg/l, 2100 mg/l, and 1700 mg/l for acetic (43 mM/l), propionic (18 mM/l), and butyric (10.5 mM/l) acids, respectively. The corresponding theoretical CODs are 2750 mg/l, 2015 mg/l, and 1680 mg/l, respectively. This can be used as design data for evaluating the economics of a selected separation process. The conductivities and the concentration of ionic species for various diets are given in Table 3. Animals consume water and the conductivities and concentration of ionic species for feed water (local water supply source) shown in the table are for comparison with the different diets. Ionic strength varied between 85 and 115 mM/l depending on the diet composition. 3.2. α-Amylase, cellulase, and total protein content Other chemicals screened for their presence in the filtered supernatant were enzymes such as ␣-amylase and cellulases. Activities of these enzymes and the total protein content for the four diets are given in Table 4 along with their typical pH values. The average pH ranged between 6.45 and 6.8 for the four diets and showed minimal variation. The average pH values for each diet in Table 4 were within ± 0.1 of the individual pH readings for that particular diet. Animals on a grain diet were fed every 2 h and animals on other diets were fed once every 24 h. The data shown is for all the diets that were collected 3.5 h after the animals were fed. The activity of cellulase was not very different for any diet, but grain-fed animals show substantially greater amounts of ␣-amylase and protein content. The quantity of feed given to each animal on different diets is identical. However, the grain-fed animal was fed at

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Table 3 Conductvity, ionic strength, and concentration of ionic species for various diets and feed water for animals Feed type

Grass

Conductivity (mS/cm) Ionic strength (mM/l) Ionic species (mg/l) K Zn Fe Mn Cu Na Ca Mg NO3 − PO4 3− Cl− SO4 2 CO3 2− HCO3 −

Grain

Alfalfa

Cobs

Feed water

8.01 101.7

8.96 113.8

7.65 97.2

6.8 86.4

0.47 6.0

1160 0.17 4.9 0.8 0.09 3000 103 46.7 0 197 278 0 0 5878

1640 0.19 1.6 0.1 0.1 2120 81.5 41.6 0 339 657 0 0 4847

1870 0.58 2.3 0.7 0.2 2760 19.9 213.4 0 157 611 0 0 5368

1085 0.1 2 0.2 0.1 1950 35 66.2 0 123 376 0 0 4587

11 0.12 0.27 0.1 0.76 43 57.2 14.4 1.1 1.2 19.5 35 0 464

regular intervals and the total quantity of feed given in a 24 h period was similar to grass, cobs, and alfalfa-fed animals. The concentrate and roughage diets make an impact on the production of protein, enzymes, and acid amounts. Commonly, the animals are given the concentrate diet such as grain. A constant supply of grain diet helps in the maintenance of concentration levels of microbes that utilize extra-cellular enzymes such as cellulase and ␣-amylase for the breakdown of the substrate. That means feeding animals at regular intervals positively impacts the production of ␣-amylase in the case of grain-feds. As seen in Table 4, higher protein content can also mean a higher enzyme concentration. Therefore, it is advantageous to provide a constant supply of grain or grass diet to the animals that in turn helps microbes release a higher concentration of enzymes for substrate breakdown. A typical meat packing house which may process 4000 animals could produce 14–142 million IU of amylase and 8–11 million IU of cellulase per day. This enormous source of chemicals can be utilized by separating these enzymes and APBs. This processing would also reduce the COD burden of wastes from slaughterhouses and provide income as a new co-product stream. Table 4 Protein content and enzyme activities in filtered rumen supernatant samples Diet

pH

Amylase (IU/l)

Cellulase (IU/l)

Protein (mg/ml)

Grain Grass Cobs Alfalfa

6.75 6.45 6.6 6.8

500 230 50 75

40 29 31 35

1.1 1.15 0.22 0.35

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3.3. Economic evaluation A meat processor spends approximately US$ 0.352 kg−1 of COD burden in a wastewater treatment plant (LWWT, 2000). The disposal cost calculations for a typical meat processing plant can be performed as shown. COD treatment cost: Number of animals per day = 4000 Average measured COD burden per kilogram of rumen fluid = 50 g/l Rumen fluid per animal = 77 l at 120 l total rumen/animal and 36% solids Total COD per day = 4000 × 77 × 50 × 10−3 = 15,400 kg COD Therefore, cost to treat the above COD = 15,400 kg BOD × US$ 0.352 kg−1 COD = US$ 5420 per day Annual (365 days) cost to treat the COD for 4000 animal operation = US$ 2,000,000 (excluding treatment for solids stream) Thus, these streams, depending on their COD burden, are an unavoidable expense to a meat processor. The annual cost of COD treatment that amounts to approximately US$ 2,000,000 can be redirected for use in process and product development (separation of VFAs and enzymes). Various process costs that include raw materials, labor costs, and other project costs can be paid from this enormous savings if product development is undertaken. The preliminary estimate of the sale of products recovered from rumen fluid in the form of calcium magnesium paunchate (CMP: conversion of volatile fatty acids to salts of calcium and magnesium using dolime), ␣-amylase and cellulase is presented below: APBs as CMP: Total quantity of APBs in supernatant = 4.75 g/l. Supernatant volume = 71 l/animal (92% of rumen fluid). Total acids = 71 × 4000 × 4.75 = 1350 kg per day. The reaction between dolime and volatile fatty acid can be represented as: CaO·MgO + 4HA + 2H2 O → Ca·MgA4 + 4H2 O. Based on the above equation and assuming 100% conversion, the amount of CMP produced is 5 kmol per day or 1740 kg per day. Assuming a selling price of US$ 1.1 kg−1 based on calcium magnesium acetate price (Cryotech Deicing Technology, Fort Madison, Iowa), the annual income from CMP sales = US$ 698,610. ␣-Amylase: Average quantity of ␣-amylase in supernatant = 250 IU/l. Total units of enzymatic activity = 250 × 71 × 4000 = 71 million IU per day Assuming 100% recovery and a sale price of US$ 5.5 per million IU, the annual income from ␣-amylase sales = US$ 142,533. Cellulase: Average quantity of cellulase in supernatant = 35 IU/l. Total units of enzymatic activity = 35 × 71 × 4000 = 10 million IU per day. Assuming 100% recovery and a sale price of US$ 75 per million IU (Sigma Chemical Co., St. Louis, MO), the annual income from cellulase sales = US$ 273,000.

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In summary, APB’s contribute an average of 36% COD to the supernatant stream, which is 16% of the rumen fluid stream. The annual savings by separating APB’s from the supernatant is US$ 320,000 (for 2464 kg per day of COD). The pellet stream carries about 57% of the ruminal fluid COD burden (8778 kg COD per day), which is equivalent to an annual disposal cost of US$ 1,140,000. Hence, excluding the pellet stream processing cost, an additional annual saving of US$ 1,140,000 is realized. Thus, product separation and recovery from ruminal fluid can be an attractive alternative that not only reduces the COD costs, but can also bring profits from the saleable recovered products (a total of US$ 1,114,143, i.e., US$ 698,610 from CMP, US$ 142,533 from ␣-amylase, and US$ 273,000 from cellulase). 3.4. Potential recovery processes Waste streams can be treated using a wide variety of separation methods. Products and/or waste streams can be marketed or safely discharged after the treatment. Adsorption, extraction, and precipitation can be considered toward the recovery of acids and enzymes. These processes can be used on the bulk stream to affect preliminary separation, and later specific methods such as chromatography and ultrafiltration can be used to further enhance the recovery. Adsorbents have been developed for many different industrial separations. Some of the commonly used adsorbents are activated carbon, activated alumina, silica gel, and molecular sieves zeolites. A large surface area per unit weight of the adsorbent along with their surface selectivity due to hydrophobic or hydrophilic sites can be used to separate both acids and proteins. Instances can be found in the literature where the adsorbents mentioned above have been used for the separation of acids or enzymes (Ganguly and Goswami, 1996; Gupta et al., 1994). The minimum requirement for liquid extraction is the intimate contact of two immiscible liquids for the purpose of mass transfer of constituents from one liquid phase to the other, followed by physical separation of the two immiscible liquids. The distribution of a substance between two phases is described by a partition coefficient. Literature shows the extraction of acids (Eyal and Canari, 1995) and enzymes (Chang and Chen, 1995) with amine based extractants. A separation approach that may contribute to lower operating and utility costs is a strategy in which acids and enzymes are recovered simultaneously. There has been no documentation in the literature either on the recovery of chemicals from a ruminal stream or on the simultaneous separation of acids and enzymes. The latter is a novel approach that can increase the overall economic feasibility of recovering chemicals from this stream. Using extraction and adsorption, the volatile fatty acids and enzymes were simultaneously separated from the aqueous stream (Clements and Koppolu, 2002; Koppolu, 2002). Precipitation is most commonly employed in the early part of the separation process to reduce volume. It is carried out by the addition of a salt. For example, the volatile fatty acids can be precipitated using oxides of calcium or magnesium or zinc (Clements and Koppolu, 2002), and enzymes or proteins are routinely separated by this method in biological operations (Wheelwright, 1991). Separation processes using membranes work by selectively allowing one or more components from a stream through a membrane. Varieties of membrane processes are available

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with different membrane characteristics. Ultrafiltration can be used where the separation is based on particle size and molecular diffusion principles. This method can be used to concentrate the rumen fluid to separate small inorganic and organic molecules from the protein components, thus enriching both streams. The separation of organic molecules and proteins from fermentation broths is carried out on a regular basis (van Reis and Zydney, 2001). Ultrafiltration has a few advantages over other concentration methods, such as the non-requirement of chemicals, concentration without phase change, and simple materials of construction. The separation of volatile fatty acids and enzymes from ruminal stream using ultrafiltration membranes can be found (Koppolu, 2002). Many types of chromatography are being used in the separation and purification of acids and proteins. However, chromatography of proteins using underivatized adsorbents such as silica gel in the column mode is a cheaper approach. The column process becomes more cost effective if the packing material is a bare adsorbent without any surface modifications. The cost of the resin with surface modifications is a major expense, and efforts must be expended to maintain its suitability for use. Even if the packing material is discarded after a few cycles, the operation could become prohibitively expensive, since the underivatized adsorbent is cheap when compared to the modified surface packing that is commonly used. Literature discusses the separation of enzymes or acids using different modes of chromatography (Karger and Hancock, 1996; Teunissen et al., 1993). Recovery processes become cost effective if the operating steps are simple and chemicals used to separate solutes from solution are relatively cheap. Studies can be carried out through model or/and ruminal solutions to gather information toward building process development schemes. The important factor to consider during the course of process development is the activity or the concentration of a particular solute. The activity should not decrease nor should it be destroyed. 4. Conclusions The total amount of VFAs and the individual weight ratios of each acid are consistent with the values reported in the literature. The high values of COD in rumen samples suggest a burden that has to be met in effluent treatment. Characterization of rumen samples in the manner described in this work has revealed substantial amounts of “hidden” chemicals, which can be potentially recovered as an economic asset. Thus, the value-added chemicals like VFAs and enzymes (such as ␣-amylase and cellulases) can be tapped if a continuous process is devised such that it is compatible with the current meat processing operation. Therefore, a serious effluent problem like rumen waste which requires treatment for COD can be transformed into a cash flow to the meat processor through an economical process development to recover useful products. References APHA. Standard methods for the examination of water and wastewater, vol. 15. Washington, DC, 1980. Barnett AJG, Reid RL. Reactions in the rumen. London: Arnold publishers; 1961. Bergman EN. Energy distributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol Rev 1990;70(5):567–90.

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