Single-Cell Protein Production by Photosynthetic Bacteria

Single-Cell Protein Production by Photosynthetic Bacteria

Single-Cell Protein Production by Photosynthetic Bacteria R. H. SHIPMAN,L. T. FAN,AND I. C . KAol Department of Chemical Engineering, Kansas State Uni...

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Single-Cell Protein Production by Photosynthetic Bacteria R. H. SHIPMAN,L. T. FAN,AND I. C . KAol Department of Chemical Engineering, Kansas State University, Manhattan, Kansas I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Process Considerations ....... 111. Conceptual Design ..................................... IV. Economic Analysis ........................... A. Capital Investm ........................... B. Annual Operating Costs ............................. C. Total Production Cost and Profitability . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

161 166 172 176 176 178 179 181

I. Introduction Photosynthesis is one of the basic biochemical processes, in which plants, algae, and specialized populations of bacteria convert the energy from sunlight or solar energy into chemical energy for cellular biosynthesis. Man has used this natural process of harnessing solar energy in the development of algal cultivation systems for secondary waste treatment and for the production of human foods, livestock feeds, and fertilizers. Although previous investigators (Hirayama, 1968; Wai, 1971, 1972; Thanii and Simard, 1973; Shipman, 1974a; Shipman et al., 1975) have suggested that photosynthetic bacteria may be a potential feed source for aquaculture systems or livestock, processes for mass-culturing photosynthetic bacteria as a source of human food are undeveloped at this time. Photosynthesis, as it occurs in plants and algae, differs from that of photosynthetic bacteria in several respects as diagrammed in Fig. 1. Plant photosynthesis is basically an aerobic process in which CO, is used as the sole carbon source for cellular biosynthesis. Plants and algae contain different photosynthetic pigments, or chlorophylls, and are capable of hydrolyzing water to derive reducing power (H+)for biosynthesis and molecular oxygen. Bacterial photosyntheses are anaerobic processes in which molecular hydrogen, reduced sulfur compounds, and organic compounds are used as exogenous electron donors. Bacteriochlorophylls, the photosynthetic pigments of photosynthetic bacteria, play a role similar to that of algal chlorophylls. Some photosynthetic bacteria, like plants, may use COz as the sole carbon source for cellular synthesis; however, the overall process requires an inorganic chemical reductant, such as H, or H,S. Other photosynthetic bacteria use light energy for the conversion of organic compounds to cell material. Water is never used as the ultimate electron donor; this 'Biochemical Development Division, Eli Lilly and Company, Indianapolis, Indiana.

161

162

R. H. SHIPMAN, L. T. FAN, AND I. C.

KAO

Green plants and algae

Photosynthetic bacteria

Light

Light

Chlorophyll

0 2

organic compounds

NADPH

+

FIG. 1. Comparison of plant and bacterial photosynthesis. Adapted from Pelzar and Reid (1965).

explains why molecular oxygen is never produced as an end product of bacterial photosynthesis (Rose, 1968).The basic photosynthetic reactions of algae and bacteria are depicted in Table I. The pigment system of photosynthetic bacteria plays an important lightgathering function in the assimilation of light energy. This pigment system consists of various bacteriochlorophylls, which differ in absorption spectra, chemicaI composition, and structure, and a number of identical or closely related carotenoid pigments. Bacteriochlorophyll excitation occurs when a bacteriochlorophyll molecule absorbs a quantum of light resulting in the loss of an electron, i.e., BChl [email protected] BChl+

+ e-

The released electron then migrates through the photosynthetic unit to transfer its energy to a special reaction-center chlorophyll. In photosynthetic bacteria, electrons flow through a series of intermediates in a specialized transport system, depicted in Fig. 2, with electrons migrating through an unknown intermediate, to ferredoxin, ubiquinone, cytochrome b, and cytochromef terminating with the return of BChl+ to a “ground state.” In the intermediate step between cytochromes b andf, synthesis of adenosine triphosphate (ATP) occurs. This cyclic process, by which solar energy is biotransformed into cellular chemical energy, is designated “cyclic photophosphorylation.” Carotenoid pigments are important participants in the photosynthetic process, where they function primarily as light-harvesting pigments responsible for transmitting absorbed light energy to bacteriochlorophyll. The ef-

163

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

TABLE I BASICPHOTOSYNTHETIC REACTIONSOF PLANTSAND MICROORGANISMS~

I.

Higher plants and unicellular algae

COZ

+ H20

solar energy

(CHZO) +

____)

0 2

chlorophyU

biomass

11. Certain algae and bacteria

CO,

+ 2H,

solar a e r g y

(CHZO)

+ HZO

(CH,O)

+ S + H,O

chlorophyll or

bacteriochlomphyll

111.

Photosynthetic sulfur bacteria

CO,

+ H,S

solar energy ____2)

bacteriachlorophyll

IV. Purple photosynthetic bacteria

CO,

+ CHO + HZO orgaluc

material

solar energy

(CHZO) + Hz

-w

+ HzO

bacteriachlorophyll

~~

~

~

-~

=Adapted from Stanier et ul. (1970).

fectivenessof energy transfer from the carotenoids to the bacteriochlorophyll has been estimated from 3&50% up to 90% in certain species of Rhodopseudomows (Sybesma, 1970). Carotenoid pigments also have an important function in phototaxis by purple photosynthetic bacteria (Clayton, 1953). Finally, a more fundamental function of carotenoid pigments is the protection of light-sensitive bacteriochlorophylls from the harmful photooxidizing effects of bright light. Photosynthetic bacteria are widespread in nature and have been encountered in almost every body of water and in the soil. They are especially abundant in stagnant waters containing decaying organic matter or other organic substances. In lakes and ponds, masses of photosynthetic bacteria frequently develop as stratified layers between water layers containing oxygen and those layers containing H2S. The development of photosynthetic

164

R. H. SHIPMAN, L. T. FAN, AN D I . C. KAO

*_--

__---

,/-

,'

,' Unknown ' Intermediate I

I

/

:

H h e d o x in I

fI I

I

Ubiquinone I

1

Cy!ochrome b

'.

t

0-

,

I

\\

Cyifchrome f

\

Light at 880 nrn (far red)

'.

FIG. 2. Cyclic photophosphorylation in photosynthetic bacteria. Adapted from Brock (1970).

bacteria depends, as a rule, on the presence of light, the concentration of H2S, and adequate anaerobic conditions (Kondrat'eva, 1965). Photosynthetic bacteria are represented by a rather large number of species differing in morphology, pigmentation, physiological, and biochemical properties. Some of the important physiological, biochemical, and morphological properties of the three major groups of photosynthetic bacteria are shown in Table 11. The purple sulfur bacteria, Thiorhodaceae, are strict anaerobes, photoautotrophic, and are able to oxidize H2S and other organic compounds. Purple sulhr bacteria accumulate droplets of elemental sulfur within their cytoplasm during the initial step of H2S oxidation. The intracytoplasmic sulfur deposits disappear in the second step with the oxidation of sulfur to sulfates and the disappearance of H2S from the cellular environment. The green sulfur bacteria, Chlorobacteriaceae, are easily distinguished fiom the

TABLE I1 MORPHOLOGICAL, PHYSIOLOGICAL, AND BIOCHEMICAL PROPERTIES OF OF PHOTOSYNTHETIC BACTERIA'

THE

THREE MAJORFAMILIES

Green sulfur bacteria (Chlorobacteriaceae)

Purple sulfur bacteria (Thiorhodaceae)

Nonsulfur purple bacteria (Athiorhodaceae)

Major pigment system

BChl c, abs. max. 747 nm BChl d, abs. max. 725 nm

BChl a, abs. max. 820 nm BChl b, abs. max. 1025 nm

BChl a, abs. max. 820 nm BChl b, abs. m a . 1025 nm

Cell morphology

Motile or nonmotile rods, cocci, pleomorphic; some with gas vacuoles

Motile or nonmotile rods, cocci, pleomorphic; some with gas vacuoles

Motile rods or spirals; some multiply by budding

Photosynthetic electron donors

H,S, thiosulfate, H, (organic compounds by some strains)

HzS, thiosulfate, H, (organic compounds by some strains)

HZrorganic compounds (HzS usually toxic)

Sulfur deposition

Always outside the cell

Usually inside the cell, except Ectothiorhodaceae

None

Aerobiosis

Obligate anaerobes

Obligate anaerobes

Facultative; grow in the dark aerobically, with photosynthetic growth anaerobically

Growth factor requirements

B12or none

Blz or none

Usually complex

DNA base composition (I G = C)

48-58

46-67

61-73

Property

~

"Adapted from Pfenning (1967).

A

r

g C

3 6

m .c

I66

R. H. SHIPMAN, L. T. FAN, AND I. C. KAO

purple photosynthetic bacteria by their pigmentation. They are photoautotrophic, strictly anaerobic, and also capable of oxidizing H,S and other sulfur compounds. The purple nonsulfur bacteria, Athiorhodaceae, are photoheterotrophic bacteria which use organic compounds as electron donors and as carbon sources; they may also require specific factors for growth. Although these organisms can reduce CO,, they derive most of their cellular material from organic nutrients. A common organic substrate for photosynthesis by nonsulfur purple bacteria is acetic acid. Under anaerobic conditions in the light, almost 90% of the assimilated carbon in the organic substrate is converted into the intracellular reserve material, poly-P-hydroxybutyric acid while only some 10% is oxidized to CO, (Stanier, 1961). Certain species of the nonsulfur bacteria are facultatively aerobic and are capable of developing nonphotosynthetically in the absence of light (Van Niel, 1944). This work is concerned with examination of the technical and economic feasibility of the bacterial photosynthetic process for producing single-cell protein (SCP) from agricultural by-products and wastes.

It. Process Considetations The basic design parameters have been extrapolated from experimental data generated by the present authors (Shipman, 1974a; Shipman et al., 1975) and by Kobayashi et al. (1970). Additional parameters that must be considered in the photosynthetic design are: (1)nutrient source, (2) generation or doubling time, (3)effects of oxygen on pigment synthesis and photosynthetic growth, (4) pH, and (5) optimum temperature. Photosynthetic bacteria may be grown on media of known and simple composition. A number of experimental culture media have been described for the cultivation of photosynthetic bacteria (Hutner, 1950; Sistrom, 1960; Ormerod et al., 1961; Bose, 1963);however, various agricultural by-products may be substituted as an economical source of nutrients. Purple photosynthetic bacteria are frequently among predominating populations of microorganisms in ponds, ditches, and other water sources polluted by sewage and other types of organic matter. Thus cultivating photosynthetic bacteria on a mass-scale in food processing wastes or other agricultural by-products also appears to be a reasonable alternative. Previous studies (Wai, 1971, 1972)have indicated that photosynthetic bacteria may be profusely cultured in extracts of crude carbohydrates prepared from bananas, potato starch wastes, wheat bran, and rice bran. In this and other related works, the use of an infusion prepared from the acid-hydrolysis of wheat bran, containing primarily hydrolyzed wheat starch and proteins, is considered as the nutrient source for photosynthetic cultivation (Shipman, 1974a; Shipman et al., 1975).

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

167

The growth of purple photosynthetic bacteria has been reported on media containing ammonium salts, nitrates, urea, various amino acids, peptones, or yeast autolyzates as nitrogen and carbon sources (Van Niel, 1944; Gest and Kamen, 1949; Cohen-Bazire et al., 1957). In the presence of light and under absolute anaerobic conditions, purple photosynthetic bacteria are able to fix atmospheric nitrogen (Kamen and Gest, 1949). In an atmosphere exceeding 4% oxygen, nitrogen fixation may be completely suppressed (Pratt and Frenkel, 1959). Nitrogen fixation is inhibited by the presence of ammonium salts in the medium and is reversibly inhibited by molecular hydrogen in concentrations exceeding 60% (Gest and Kamen, 1949). Purple bacteria can fix molecular N2 both in organic and mineral media. Investigationsby Gest et al. (1950), have shown that N+S stimulates nitrogen fixation by Rhodospirillum rubrum grown on media containing organic compounds. Studies of nitrogen fixation in an atmosphere enriched with 15N have revealed that the distribution of this isotope in photosynthetic bacterial cells is similar to the distribution of I5N in the cells of Azotobacter. The largest amounts of intracellular I5N have been detected in the glutamic acid and the ammonium fractions (Wagenknecht and Burris, 1950). The vitamin requirements of photosynthetic bacteria may vary considerably. Except for vitamin BIZ, most purple sulfur bacteria do not require vitamins; thus, the supplementation of the culture medium with various vitamins does not result in stimulation of growth (Kondrat’eva, 1965). The addition of some B vitamins is required for the growth of typical representatives of nonsulfur purple bacteria (Hutner, 1944, 1950), and the specific requirement for a particular vitamin may be a distinguishing characteristic for certain bacterial strains, as shown in Table 111. Bacterial photosynthesis is basically an anaerobic process in which the presence of sunlight is an indispensable condition; however, under certain environmental or culture conditions, several species of photosynthetic bacteria may be grown aerobically. Several species of Rhodopseudomonas and Rhodospirillum are facultative aerobes and can grow in the presence of oxygen (Van Niel, 1944; Gest, 1951). All other representatives of the purple photosynthetic bacteria, including the nonsulfur purple bacteria, are obligate anaerobes. Purple nonsulfur bacteria that grow with organic substrates in the dark utilize a purely respiratory metabolism, using the Krebs cycle as a pathway of terminal substrate oxidation, while under anaerobic conditions in the light, an anaerobic light-dependent Krebs cycle is functional (Eisenberg, 1953; Kondrat’eva, 1965). Cultures of photosynthetic bacteria grow rapidly when they are illuminated under anaerobic conditions. In a study examining the effects of 0, on pigment synthesis (Lascelles and Wertlieb, 1971), the generation time of photosynthetic bacteria incubated anaerobically in the light has been found to be ofthe order of 2.5-3 hours, whereas generation times of mutant strains

168

R. H. SHIPMAN, L. T. FAN, AND I. C. KAO

TABLE I11 NONSULFURPURPLE BACTERIA"

VITAMIN REQUIREMENTS OF

Thiamine

+ +

biotin Thiamine

+

Biotin

p-Aminobenzoic acid

Thiamine

biotin

nicotinic acid

Rhodospirillum rubrum

+

-

-

-

-

Rhodopseudomonas palustris

-

+b

-

-

-

Rhodopseudomonas capsulatus

-

-

+

-

-

-

Microorganism

Rhodopseudomonas gelatinosa

-

-

-

+

Rhodopseudomonas spheroides

-

-

-

-

+

'Adapted from Hutner (1946). bFolic acid may be substituted for p-aminobenzoic acid (Hutner and Scher, 1961).

incubated aerobically in the dark varied from 6 to 10 hours. The atmospheric oxygen content is also an important factor in the synthesis and cellular content of the bacteriochlorophylls and carotenoid pigments. The bacteriochlorophyll concentration in purple photosynthetic bacteria normally varies from traces to 25% of the cellular dry weight (Lascelles, 1963). In addition to oxygen concentration, such parameters as light intensity and the relative age of the culture may influence the bacteriochlorophyll content. The greatest concentration of intracellular bacteriochlorophyll may be found during the exponential growth phase. An elevated oxygen tension exerts a unique control over pigment synthesis, as demonstrated by the inhibition of bacteriochlorophyll and carotenoid synthesis in cultures grown aerobically in the dark (Cohen-Bazire, 1963). Introduction of 0, into growing, illuminated cultures will also rapidly inhibit further pigment biosynthesis; however, when illuminated cultures are returned to anaerobic conditions, bacteriochlorophyll synthesis is restored. The repression of pigment synthesis by 4 rather than by an obligatory requirement for light has been suggested as a possible mechanism for the absence of bacteriochlorophyll in nonilluminated, aerobically grown cultures of photosynthetic bacteria (Lascelles, 1960). This theory is supported by the fict that if the atmospheric oxygen content is lowered to 1-6%, photosynthetic bacteria can synthesize bacteriochlorophyll and carotenoids in the dark. Oxygen markedly influences carotenoid pigment synthesis by photosynthetic bacterial cells. Carotenoid synthesis by photosynthetic cells culti-

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

169

vated in light and darkness is almost totally suppressed by vigorous aeration (Goodwin, 1955). Exposure to light also leads to quantitative changes in the cellular carotenoid content; as light intensity increases, carotenoid synthesis decreases. The exact mechanism by which photosynthetic bacteria control pigment biosynthesis is unknown. It may seem paradoxical that photosynthetic pigments, which are formed in the light, are found in greater concentration in bacterial cells grown in dim light than in cells grown in bright light. The ecological explanation for this phenomenon is that in dim light it is of advantage to the cell to have a large amount of light-gathering pigment in order that more of the light reaching the cell can be captured and transformed into chemical energy. When growing aerobically in the dark on organic compounds, pigment synthesis is repressed so that energy made available by oxidative reactions is not diverted into unnecessary materials. Photosynthetic bacteria frequently occur in natural habitats that are reached only by a small percentage of the solar radiation. Although the presence of light is an indispensable condition for photosynthetic growth, illumination is not optional for the bacteria in their natural habitats insofar as their light requirements are concerned. It is not that photosynthetic bacteria prefer environments of low light intensity, but rather that they have adapted to such environments. In nature, purple photosynthetic bacteria can develop under a layer of algae because of the differences in their absorption spectra. While photosynthesis in green plants and algae utilizes light waves mainly in the range of 700 nm, purple bacteria perform photosynthesis by absorbing light waves in the range of 800-900 nm (Kondrat’eva, 1965). Purple photosynthetic bacteria absorb light primarily in the infrared zone of the spectrum, and in some instances light filters (for wavelengths greater than 800 nm) may be used for selectively cultivating photosynthetic bacteria (Van Niel, 1944). Whenever possible, purple photosynthetic bacteria attempt to grow close to the surfice where they are often found at a depth of 20-50 cm (Kondrat’eva, 1965). Several investigators have examined the effects of light intensity on growth rate, cell production, pigment biosynthesis, and C 0 2 assimilation. In studies by Sistrom (1962), the effects of light intensity on growth rate and pigment synthesis have been examined (Fig. 3). The studies indicate that a decrease in light intensity not only results in an increase in pigment content, but also leads to a decrease in growth rate. The maximum specific growth rate has been reported to be approximately 0.75 doubling per 100 minutes, corresponding to a generation time of slightly greater than 2 hours. Other studies have revealed that light intensities below 7 x 103 erg.cm2/ sec are strongly inhibitory to photosynthetic bacterial growth in mineral media (Kondrat’eva, 1965), and that an increase in the light intensity from 7

170

R. H. SHIPMAN, L. T. FAN, AND I. C. KAO

OL

o,:

1000

2000

3000

4000

1 5000

Light Intensity (foot candles)

FIG. 3. The specific growth rate constant and the specific bacteriochlorophyll content of Rhodopseudomonas sphermdes at various light intensities. Adapted from Sistrom (1962). 0-- -0,Growth rate;-., chlorophyll protein.

X 103 to 12 to 15 x 103 erg.cm2/sec markedly stimulates cell growth; however, light saturation ensues at light intensities exceeding 15 x 103 erg * cm2/sec. Photosynthetic growth is affected differently in media containing organic compounds, such as acetic acid, propionic acid, or glucose. In such media, rapid bacterial growth proceeds at light intensities of 2 to 3 x 103 erg cm2/sec. The cell multiplication rate increases with light intensity to an approximate value of 7 x 103 erg.cm2/sec; at light intensities exceeding 12 x 103 erg -cm2/sec, photosynthetic bacterial growth is inhibited (Kondrat'eva, 1965). This phenomenon may be explained by the fact that at low light intensities photosynthetic bacteria behave photoheterotrophically by metabolizing organic compounds. At a higher light intensity, they grow photoautotrophically, biosynthesizing their cellular compounds from CO,. Studies by Hirayama (1968)have shown that illumination of 7ooO-10,oOO lux (650-930 lumens/ft2)is sufficient for normal lipid and pigment synthesis by photosynthetic bacteria. Heden and Levin (1959)have grown photosynthetic bacteria within closed stainless steel fermenters by means of internal illumination. A light intensity of 100 lumensfliter in preliminary glass-flask cultivations has been found to be optimal, higher light intensities actually depressing growth. It has also been found that illumination intensities of 120 lumediter in 1000-liter

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

171

fermenters and 30.0 lumen/liter in 3000-liter fermenters, deviate only slightly from the optimum obtained in glass-flask cultivations. Photosynthetic bacteria have been reported to grow within a wide range of temperatures. Maximum growth and COz reduction occur within a range of 30"40"C (Katz et al., 1942). Van Niel(l944) has indicated that the optimum temperature for most photosynthetic bacterial growth is approximately 37°C. The present authors (Shipman, 1974a; Shipman et al., 1975)have obtained a generation time of 4 hours for photosynthetic growth of Rhodopseudomonas gelatinosa at 35°C. Low temperatures may have deleterious effects on photosynthetic growth. In a study by Dworkin (1959), low-temperature (1°C) cultivation of photosynthetic bacteria led to the destruction of bacteriochlorophyll and subsequent death of bacterial cells. Dworkin concluded that carotenoids lose their protective function at low temperatures as a result of the inactivation of the enzyme system participating in their reduction. The pH and H2S concentration are very important for the predominant development for many forms of purple photosynthetic bacteria. The pH range for the growth of photosynthetic bacteria and the optimal pH for various species of bacteria depend not only on the HzS concentration, which should not exceed 150-200 mg/liter, but also on the presence of other organic and inorganic compounds in the medium and their respective concentrations (Van Hiel, 1931). The range of the optimal pH for various strains and species of photosynthetic bacteria is very wide. The optimum medium pH for most of them range between 7.0 and 8.5; only for a limited number of photosynthetic bacteria does the optimum fall between 6.5 and 6.8 (Pfenning, 1967). Depending on the composition, the culture medium may become more acidic or more alkaline or the pH may remain unchanged during the photosynthetic bacterial growth. Changes in the pH of various media during photosynthetic growth are normally associated with: (1)the assimilation or release of CO,; (2) the production of sulfuric acid by the oxidation of HzS or thiosulfate; or (3)by the accumulation of organic acids in the medium. These characteristic changes in pH normally associated with the formation of primary products of photosynthesis by purple photosynthetic bacteria are well documented in the literature (Kondrat'eva, 1965). In some cases, the pH changes in the medium are indicative of the utilization rate of organic acids (i.e., acetic and propionic acids) since each mole of organic acid utilized induces a definite change in the pH of the medium (Clayton, 1955). Studies of the mineral requirements of photosynthetic bacteria have established the need for sodium, potassium, calcium, cobalt, magnesium, and iron (Hutner, 1944, 1946). In nature, photosynthetic bacteria are encountered both in freshwater and saline reservoirs, including bodies of water

172

R. H . SHIPMAN, L. T. FAN, AND I. C . KAO

having high salt concentrations. Photosynthetic bacteria isolated from very salty water require 10-15% NaCl in the growth medium (Van Niel, 1931). A concentration of 24% NaCl has been found to be optimal for most purple bacteria originating from various saltwater sources. Purple photosynthetic bacteria isolated from freshwater reservoirs require small amounts of sodium which may be supplied by 0.1-0.2% concentrations of NaCl in the medium (Kondrat’eva, 1965). Potassium requirements of photosynthetic bacteria are on the order of 5.0 mg/liter. Photosynthetic growth may be inhibited when the K:Na ratio exceeds 5, regardless of the absolute concentrations of K and Na in the medium. Purple photosynthetic bacteria have been reported as being very sensitive to calcium deficiency-undergoing agglutination in its absence (Hutner, 1946). The Ca requirements of purple bacteria may be satisfied by the addition of 0.001-0.005% CaClz. Certain photosynthetic bacteria require cobalt in concentrations of 5 pg/lOO ml for the synthesis of cobalamin (BI2) (Kondrat’eva, 1965). Magnesium is required by all photosynthetic bacteria for the biosynthesis of bacteriochlorophyll. Magnesium also participates with various other metals in the activation of several enzyme systems (Van Niel, 1931). The growth of photosynthetic bacteria and C02 fixation may be stimulated by the addition of small quantities of Mn to the media. Quantities of 0.5 pg of Mn per 100 ml may be sufficient for optimum growth; however, higher concentrations of Mn, up to 10 pg/100 ml, have neither stimulatory nor inhibitory effects on growth (Kondrat’eva, 196s). Iron is an indispensable constituent in the medium for the synthesis of iron porphyrins and other iron compounds by purple photosynthetic bacteria (Lascelles, 1953). The important function that cytochrome performs in the metabolism of purple bacteria is well documented. Iron deficiency inhibits the growth of photosynthetic bacteria and markedly suppresses bacteriochlorophyll synthesis (Kamen, 1955).

Ill. Conceptual Design Here, the proposed photosynthetic SCP process, illustrated in Fig. 4,has been designed as a semicontinuousprocess with a mean production capacity of 5 tons of SCP per day. The daily raw material balance data are shown in Table IV. The design is based on the following cultivation parameters: (1) a digester retention time of 24 hours; (2) an operating temperature of 37°C; (3) a pH of 7.0-7.2; and (4) a harvested cellular yield of 10.0 gm of dried cells per liter. The major processing steps include hydrolysis of the wheat bran slurry, solids removal, neutralization, photosynthetic cultivation, recovery, and purification. Major pieces of process equipment and their costs are itemized in Table V.

173

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA Hydrochloric Acid (36%)

1.

Hydrolysir - Sterilization

Solids Removal

Bran Wheal Bran Intu8ian

'sunlight/lncandescent

II

Photosynthetic

Solids Recovery

Wheat Bran Residue

Lighting

lnoculatim

Anhydrous Ammonia Yeast Extract

I Supernatant Recycle

H ar ye s t ed ~:/,osynthelic

Centrifugation

Water

FIG. 4. Flow sheet for the photosynthetic single-cell protein process.

In the initial processing step, an aqueous slurry of wheat bran, containing approximately 30% solids, is acidified with HC1(36%). The slurry is injected with steam and held at a temperature of 121°C and a steam pressure of 15 psi for 4 hours. The unhydrolyzed wheat bran residue is separated from the supernatant in a solid-bowl, conveyor-type centrifuge. The wheat bran infusion is discharged into a stainless-steel tank in which neutralization with TABLE IV DAILYRAW MATERIAL BALANCE FOR THE PHOTOSYNTHETIC SINGLE-CELL PROTEIN (SCP) PROCESS Component Input Wheat bran HCI (36%) Anhydrous ammonia Yeast extract Makeup water output Photosynthetic SCP (dry weight) Wheat bran residue (85-90% solids)

Tonslday

15.0 4.7 0.675 0.099 470.0 5.0 6.75

TABLE V MAJOR PROCESS EQUIPMENT FOR PHOTOSYNTHETIC SINGLE-CELL PROTEIN(SCP) PRODUCTION Unit Mixing and storage tanks Mixing and storage tank Storage tank Centrifuge Horizontal transparent polyvinyl chloride (PVC) pipe Centrifuge Incandescent lamps Boiler unit

No.

2 1 1 1 33

Description

Unit cost

Cost

Type: stainless steel; capacity: 400,000gal Type: carbon steel; capacity: 24,000gal Type: carbon steel; capacity: 12,000gal Type: solid bowl with conveyor discharge; stainless steel; 90 GPM hydraulic capacity Dimensions: 61 cm X 67.1 m; capacity:

$33,[email protected]

$f%000

4700 gal

1 200 1

Pump

1

Pumps

5

Spray dryer

1

Type: disk bowl with nozzle discharge; stainless steel; includes drive and controls; 90 GPM hydraulic capacity 400 Watts; 115 volts Steam capacity: 30,000Ib/hr; steam pressure: 250 psi Type: radial-flow centrifugal; stainless steel; 10 HP max. capacity: 150 GPM Type: radial-flow Centrifugal; carbon steel; 10 HP max. capacity: 150 GPM Evaporative rate: 4 tons/hr

$11,200 $8,400 $50,000

$11,200 $8,400 $50,000

$22.501

$163,800

$32,400

$32,400

$6.00 $52,000

$1,200 $52,000

$5,380

$5,380

$1,680

$8,400

$26,400

$26,400

linear ft

Total purchased equipment cost:

$425,180

P x

n

E0

TABLE VI COMPOSITION OF WHEAT

Proximate analysis Ash Crude fiber Ether extract N-free extract Protein (N X 6.25)

BRAN"

% Dry basis

6.9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 4.6 .............................. 59.3 .............................. Vitamins 18.0 . . . . . . Amino acids (%) Choline Arginine 1.12 Folic acid Cystine 0.34 Niacin Glycine 1.01 Pantothenic acid Histidine 0.34 Riboflavin Isoleucine 0.67 Thiamine Leucine 1.01 a-Tocopherol Lysine 0.67 Methionine 0.11 Phenylalanine 0.56 Threonine 0.45 Tryptophan 0.34 Tyrosine 0.45 Valine 0.79

=Adapted from Jurgens (1973).

}

Minerals Calcium Iron (mdkg) 1,110.0 2.0 235.1 32.6 3.5 8.9 12.1

Magnesium Phosphorus Potassium Sodium Cobalt Copper Manganese

(%)

0.16 0.02 0.62 1.32 1.39 0.07

( d1.10 w

m

13.80 130.00

m

1 E

176

R. H. SHIPMAN, L. T. FAN, A N D I. C. KAO

anhydrous ammonia occurs. Although wheat bran contains significant amounts of amino acids, vitamins, and minerals (Table VI), yeast extract is added to supplement the infusion medium with vitamins that may have been destroyed during the hydrolysis and heating process. The temperature of the infusion medium is lowered to approximately 40°C at this stage of the process by pumping cooling water from the temperature controller of the photosynthetic cultivation tanks through the cooling jacket of the neutralization tank. Hot water flowing from the cooling jacket is then pumped back to the temperature controller and is used to maintain a temperature of 37°C within the photosynthetic bioreactor. Sterilization of the infusion medium simultaneously occurs during the acid-hydrolysis and heating process, and thus the need for an additional medium-sterilization step is eliminated. During the photosynthetic cultivation phase, wheat bran infusion is pumped from the holding tank at a rate of 5OOO GPH (18,900 Yhour) into a series of horizontal photosynthetic cultivation tanks, 61.0 cm in diameter, constructed from transparent polyvinyl chloride (PVC). Direct sunlight provides illumination for daytime photosynthetic cultivation, and 400-watt incandescent lamps, which may be lighted by solar energy stored during daytime, provide artificial illumination for nighttime operation. The retention time in the transparent PVC cultivation tanks is 24 hours. Continuous inoculation of photosynthetic bacteria is provided by recycling a portion (2%) of the exit stream at a rate of 100 GPH. Photosynthetic bacterial cells are recovered from the exit stream by pumping spent culture media from the recovery tank to a disk bowl-type centrifuge. Harvested bacterial cells are washed with water and the final product is dried in a spray dryer. IV. Economic Analysis The economic feasibility analysis of the photosynthetic SCP process given here is based on the production capacity of the previously described conceptual process. Cost estimates have been compiled from available data on similar items of equipment, cost indexes, and available cost-capacity factors. All estimates have been escalated to the last quarter of 1975 according to the Marshal Stevens Index.

A. CAPITALINVESTMENT The purchase costs for major pieces of photosynthetic SCP processing equipment are included in Table V. The base cost estimates for the stainless steel acid-hydrolysis and neutralization tanks have been taken from Guthrie (1969) and include delivery costs but not installation costs. The process centrifugation costs and their hydraulic capacities have been extrapolated

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

177

from similar items of equipment used for solids removal and dewatering of wastewater sludges (Liptak, 1974). The photosynthetic reactor used in the study is of similar design to a photosynthetic bioreactor constructed of transparent acryl resin (Kobayashiet al., 1971). Since costs for the construction of such reactors are not available, the base cost of the photosynthetic reactor has been estimated from the cost of transparent PVC pipe at $22.50 per linear foot. Costs for steam generation equipment, spray dryers, and additional processing equipment have been obtained from other sources (Perry, 1963; Peters and Timmerhause, 1968; Popper, 1970). Total purchased equipment costs including delivery amount to $425,180. Other direct costs and indirect costs, summarized in Table VII, are assumed to be equivaTABLE VII ESTIMATED CAPITALINVESTMENT Processing equipment Purchased equipment costs' Installation Instrumentation and control' Piping Electricalb Buildings Yard improvements Land Total direct costs

$425,180 98,500 42,000 85,000 24000 66,500 8,000

30,000 $779,180

Indirect cost Engineering and supervisionC Construction expensed Total direct and indirect cost Contractors feese Contingency'

81,800 93,500 $954,480 47,700 95,400

Fixed capital investment (FCI) Working capital0

$1,097,580 121,950

Total capital investment (TCI)

$1,219,530 ~

"Includes delivery costs. 'Includes costs for installation. '@ 10.5% of the direct cost. d @ 12.0% of the direct cost. "@ 5% of the total direct and indirect cost. @ ' 10% of the total direct and indirect cost. "@ 10% of the total capital investment.

178

R. H. SHIPMAN, L.

T.

FAN, AND I. C. KAO

TABLE VIII

ANNUALOPERATING COSTSFOR THE PHOTOSYNTHETIC SINGLE-CELL PROTEIN PROCESS

~~~~~~

~

Direct production costs Wheat bran @ $25.00/ton HCI (36%)@ $37.00/ton Anhydrous ammonia @ $67.50/ton Yeast extract @ $ 2 W t o n NaCl @ $ 25.00/ton Makeup water @ 0.15/1000 gal. Operating labor Supervisory labor Maintenancea Operating suppliesb Fixed charges Depreciation' Taxes and insurance Financin$ Total annual operating cost

$123,750 57,390 15,040 65,340 9,280 5,580 142,560 12,000 32,900 3,300 54,880 27,440 30,490 $579,950

OMaintenance costs = 3% of fixed capital investment (FCI). *Operating supplies = 0.3% of FCI. CAnnualdepreciation of 5% of FCI. dFinancingat one-third of total capital investment at 8% for 10 years.

lent to those of a solid-liquid processing plant. Engineering and supervision costs are 10.5%of the direct costs while construction costs are 12.0% of the total direct costs. The total direct and indirect cost is $954,480. To this figure, an additional 15%to cover contractor's fees and contingency is added resulting in a fixed capital investment (FCI) of $1,097,580. Assuming that the working capital represents 10% of the total capital investment, the total capital investment amounts to $1,219,530.

B. ANNUALOPERATINGCOSTS The annual operating costs, including direct production costs, fixed charges, and plant overhead, have been determined on an annual production basis of 330 days of operation, according to standard procedures outlined by Peters and Timmerhause (1968).A breakdown of the annual operating costs is shown in Table VIII.

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

179

The costs for wheat bran have been estimated at $25.00/ton. The costs for the acid-hydrolysis and neutralization processes have been obtained from costs of similar processes (Liptak, 1974). The remaining raw material costs have been obtained from the Chemical Marketing Reporter. The operating labor requirement for continuous process supervision is based on 3 shifts per day and an average of 1.33 operators per shift. The operating labor costs are based on a rate of $4.5O/man-hour, and a yearly salary of $12,000 is provided for supervisory labor. Maintenance and operating supply costs are based on a percentage of the fixed capital investment. The photosynthetic SCP process is assumed to have an operational expectancy of 20 years with zero salvage value at the end of that time. The annual depreciation, at a yearly rate of 5% of the FCI, amounts to $54,880. Financing is estimated at one-third of the total capital investment at 8% for ten years. The total annual operating cost amounts to $579,950. C. TOTALPRODUCTION COSTAND PROFITABILITY Given the total annual operating cost and a total annual SCP production capacity of 1650 tons, the mean total production cost per ton amounts to $351.48 ($O.lS/lb or $0.39/kg). The profitability of the proposed photosynthetic process may be shown by the gross profit per ton and the gross

Unit Price of Photosynthetic SCP ($/lb)

FIG. 5. Profitability of the proposed photosynthetic process. A0 0, per ton (dollars).

- -A,

Annual (103 dollars);

I80

R. H. SHIPMAN, L. T. FAN, AND I. C. KAO

TABLE IX ESSENTIAL AMINOACID COMPOSITION OF PROTEINS FROM PHOTOSYNTHETIC BACTERIACOMPARED WITH CONVENTIONAL FOODS"

Soy protein

Amino acid

Egg protein (%)

Histidine Isoleucine Leucine Lysine Methionine PhenylaIanine Threonine Tryptophan Valine

2.4 6.6 8.8 6.4 3.1 5.8 5.0 1.6 7.4

2.4 5.4

(%I

7.7 6.3 1.3 4.3 3.9 1.4 5.2

Photosynthetic bacterial protein

(a)

3.4-3.9 4.1-4.3 7.67.9 5.6-6.0 3.0 4.34.6 2.9-4.4 -6

6.5-7.0

"Adapted from Shipman (1974a). bNot analyzed.

annual profit for various marketing prices of photosynthetic SCP illustrated in Fig. 5. At a marketing price of $0.30/lb, for example, a gross annual profit of$410,000(i.e., gross profit of$248/ton)may be realized. The photosynthetic SCP process may be economically competitive with current commercial yeast production processes, if the current marketing price of brewer's yeast at $0.45/lb is considered (Anonymous, 1975). One of the most evident advantages of the proposed photosynthetic process is the utilization of photosynthetic SCP for human food supplementation. TABLE X VITAMINCONTENTSOF PHOTOSYNTHETIC BACTERIAAND YEAST"

Vitamin

Photosynthetic bacteria (pg/100 gm dried cells)

Riboflavin (B,) Pyndoxine (Be) Folic acid Cobalamin (BI2) Ascorbic acid (C) Cholecalciferol (D3)

3,600 3,000 2,000 200 20,000 10,Ooob

"Adapted from Kobayashi (1970). bInternationalUnits.

Yeast (pg/IOOgm dried cells) 2,900 2,400 1,700 1.0

-

300,Ooob

SINGLE-CELL PROTElN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

181

Photosynthetic bacterial cells contain approximately 65%protein and significant quantities of the essential amino acids (Table IX). In addition, photosynthetic cells contain relatively large amounts of ascorbic acid, vitamin D, and various B vitamins as shown in Table X. Unlike other SCP processes based on hydrocarbons in which the possibility of substrate toxicities exist, wheat bran offers the advantage of being a readily renewable, toxic-free, natural substrate for the production of edible protein. A reduction in the total production costs may be realized by the possible reclamation and resale of the wheat bran residue as a livestock feed. Processing of the wheat bran residue would involve some form of mechanical or solar drying; however, since the marketing price for such a product is uncertain, these costs have not been considered in the present study. Another potential scheme for the production of photosynthetic SCP involves the cultivation of photosynthetic bacteria in sewage, animal manure, and feedlot wastes. A process described by Kobayashi et al. (1971) involves the cultivation of photosynthetic organisms in municipal sewage sludges and harvesting photosynthetic bacterial cells as a by-product of the purification process. Biomass harvested from photosynthetic cultivation in sewage and animal wastes could be used as a supplement for various livestock feeds (Singh and Anthony, 1968; Coe and Turk, 1973). The proposed process for producing photosynthetic bacteria as a source of edible protein shows definite economic potential. The development of photosynthetic cultivation systems utilizing sewage sludges, livestock manure, and other agricultural waste products should definitely be encouraged. ACKNOWLEDGMENT

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