Microbiology
Lipid Metabolism in Anaerobic Ecosystems Roderick I. Mackie, Ph.D., Bryan A. White, Ph.D., and Marvin P. Bryant, Ph.D.
Critical Reviews in Microbiology Downloaded from informahealthcare.com by University of California San Francisco on 12/10/14 For personal use only.
ABSTRACT In anaerobic ecosystems, acyl lipids are initially hydrolyzed by microbial lipases with the release of free fatty acids. Glycerol, galactose, choline, and other non-fatty acid components released during hydrolysis are fermented to volatile fatty acids by the fennentative bacteria. Fatty acids are not degraded further in the rumen or other parts of the digestive tract but are subjected to extensive biohydrogenation especially in the rumen. However, in environments such as sediments and waste digestors, which have long retention times, both long and short chain fatty acids are P-oxidized to acetate by a special group of bacteria, the H,-producing syntrophs. Long chain fatty acids can also be degraded by a-oxidation. Biotransformation of bile acids, cholesterol, and steroids by intestinal microorganisms is extensive. Many rumen bacteria have specific growth requirements for fatty acids such as n-valeric, iso-valeric, 2methylbutyric, and iso-butyric acids. Some species have requirements for C,, to C,, straight-chain saturated or monoenoic fatty acids for growth.
1. OVERVIEW Unlike proteins, carbohydrates, and nucleic acids, the term “lipid” covers an extremely diverse range o hemical or molecular species. The definition of lipids is th refore simple they are classed as being sparingly soluble in water but readily soluble in organic solvents such as chloroform, hydrocarbons, alcohols, ethers, and esters. Common lipids can be loosely divided into two categories according to structure. First, those structures based on longchain fatty acids or their immediate derivatives Gcluding the natural fats and oils present in plants and animals as well as phospholipids and glycolipids which have a ubiquitous distribution.’ The second category of terpenoid lipids is based on the isoprene unit and range from the simplest
7
H 2
monoterpenes to the more complex steroid and carotenoid molecules.* The terpenoids are related and characterized by their biosynthesis, in whole or in part, from rnevalonic acid. A particularly useful system of classifying lipids is the division into “neutral” or “apolar” lipids, and “polar” or “am’ phiphilic” lipids. Thus, neutral lipids w ill include simple hydrocarbons, carotenes, triacylglycerols, wax esters, sterol esters, as well as other lipids such as fatty acids, polyprenols, and sterols in which the hydrophilic function has little impact on the overall molecular characteristics. Polar lipids include phospholipids, glycolipids, sulfolipids, some sphingolipids, oxygenated carotenoids, and chlorophylls. The nomenclature of lipids recommended by IUPAC-IUB has appeared in several journals and is listed by Ratledge and Wilkin~on.~ The great diversity in lipid structure is related to their diversity in function. Lipids can act as storage materials in animal, plant, and microbial cells, where the lipids occur typically as triacylglycerols in eukaryotes and as poly-p-hydroxyalkanoate(s) in some prokaryotes. They are responsible for the structure of cell membranes occuning mainly as phospholipids. They are associated with the photosynthetic process in plants and microorganisms not only as chlorophyll, but also as quinones and pigments involved in converting light energy into chemical energy. Lipoquinones are associated with energygenerating processes. Besides their universal role in membrane structure and function, lipids also participate in the organization of bacterial cell envelopes. They occur as lipoteichoic acids associated with the cytoplasmic membrane of Gram-positive bacteria and as lipopolysaccharidesand lipoproteins in the outer membrane of Gram-negative bacteria. Extracellular lipids are also produced by both prokaryotes and eukaryotes. These may be accompanied by extracellular enzymes such as lipases, esterases, and phospholipases which aid the growth of microorganisms on external sources of fats or hydrocarbons. The pathways of lipid biosynthesis in microorganisms are similar to those in higher organisms, although the anaerobic route of unsaturated fatty acid biosynthesis is found only in b a ~ t e r i a . ~
II. INTRODUCTION The input of lipids in anaerobic environments can be substantial especially in domestic sewage and in sewage sludge. Although lipids are quantitatively important as substrates for
Roderick I. Mackie, Ph.D. (correspondingauthor), Bryan A. White, Ph.D., and Marvin P. Bryant, Ph.D., Department of Animal Sciences 458 Animal Science Lab, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801.
H
Isoprene (methylbuta-1,3-diene)
1991
449
Critical Reviews in Microbiology Downloaded from informahealthcare.com by University of California San Francisco on 12/10/14 For personal use only.
Critical Reviews In anaerobic degradation in methanogenic ecosystems they have not been studied as extensively as the degradation of carbohydrates and proteins. Many different kinds of organisms are involved in the complex process of lipid degradation and in this review the role of bacteria in this process is emphasized, although protozoa and fungi may also be i n ~ o l v e d . In ~ - gen~ eral, lipids are hydrolyzed by fermentative bacteria with the release of long- and medium-chain length free fatty acids with the glycerol and galactose moieties being fermented to volatile fatty acids, CO, and H,.5*7Fermentative bacteria are unable to oxidize the free fatty acids and this step is carried out by specialized groups of bacteria such as the H,-producing synt r o p h ~ . ~ .or ~ .some ' ~ of the acetogenic, sulfate-reducing bacteria. In the terminal stage of complete anaerobic degradation, once inorganic electron acceptors such as nitrate and sulfate are exhausted, methanogenic bacteria utilize H,, CO,, and acetate in the production of final products, CH, and C0,.*.9J2 Much of the research on anaerobic lipid degradation has focused on a single ecosystem, the rumen. In the rumen, dietary acyl lipids are first subjected to hydrolysis by microbial li~ases.".'~Free fatty acids liberated are then subjected to extensive biohydrogenation by the nunen bacteria, resulting in an enrichment of saturated fatty acids at the expense of unsaturated fatty acids. l3 Unlike sludge or anaerobic digestion in which degradation of lipids accounts for a major portion of the acetate formed, fatty acids are not degraded further in the rumen. In fact, acetate, propionate and butyrate produced during carbohydrate fermentation accumulate and are absorbed from the digestive tract and utilized as major energy sources by the ruminant animal, the mammalian partner in this mutually beneficial association. Because of the relatively short retention time of digesta in the digestive tract of the animal, the H,producing acetogens that catabolize fatty acids and the acetateutilizing methanogenic bacteria are unable to grow rapidly enough to be maintained in the system5e9except under unusual c i r c ~ m ~ t a n c eDe ~ .novo ' ~ ~ ~synthesis ~ of microbial lipids also occurs in the rumen and free acids, both saturated and unsaturated, can be incorporated into microbial cell material. As a result of these microbial transformations, rumen lipids and postruminal digesta are enriched in stearic acid (18:O) at the expense of dietary linoleic (18:2) and linolenic (18:3) a c i d ~ . ' ~ . ' ~ In this review, research on the microbiology and biochemistry of lipid degradation and synthesis in the rumen and other anaerobic ecosystems is summarized with the emphasis on research over the past 5 to 10 years. Several excellent reviews provide extensive background information and for more details of earlier work should be c o n s ~ l t e d . ~ - ~ * ' ~ * ' ~ * ' '
111. LIPID CONTENT AND COMPOSITION OF SUBSTRATES The lipid content of substrates utilized in different anaerobic ecosystems for methanogenic fermentations varies widely (Ta-
450
Volume 1
ble 1). Waste from intensively reared farm animals is often collected and used as substrate. Pig manure has the highest lipid content (8 to 19%) of the animal wastes. Domestic sewage and sewage sludge have relatively large amounts of lipids and contain a high proportion of fats, oils, and greases. Wastes from meat packing and slaughterhouses are especially high in lipids, with flotation foams containing 60 to 65% lipid on a dry basis. Generally, lipids are 75 to 95% degraded during sludge digestion and account for approximately the same amount of methane formation (20 to 30%) as the carbohydrates.L9*37*38
Table 1 Lipid Content of Substrates for Anaerobic Digestion and Dietary Lipids Consumed by Ruminants Source Cattle waste Pig waste Poultry waste Domestic refuse Domestic sewage Sewage sludge Activated sewage sludge Brewery activated sludge Abattoir activated sludge Flotation foams Slaughterhouse wastes Alfalfa, fresh Clover, red Orchard grass, fresh Ryegrass, fresh Barley, grain Brewers grains Distillers grains Corn, grain Cotton seeds Oats, grain Peanut meal soybean seeds Sunflower seed meal Wheat, grain
Lipid content (as 9% of VS or DM) 3.w.5
I . 1-19,2 1.5-2.1 6.2 10-35 8-25 5-12 10-15 15-25 60-65 25-55 2.6-3.1 2.9-5.0 3.u.9 3.2 2.1 6.5 9.8-10.3 2.24.2 23.1 5.4-6.9 6.3 18.8 8.1 1.8-2.2
Ref. 18-21 22-25 26, 27 28, 29 30-32 31, 33 34 34 34 35 35 36 36 36 36 36 36 36 36 36 36 36 36 36 36
Ruminants are herbivores and the lipids derived from forages are predominantly of leaf origin. Lipids comprise 2 to 3% of the DM of fresh forages and about 6 to 8% of the dry weight of leaf tissue. Glycolipids and phospholipids comprise approximately 95% of total leaf lipids with glycolipids comprising 70 to 80% of the complex lipids present irrespective of plant species. Most of these lipids are present in the chloroplasts of leaf tissue. Chloroplasts contain 22 to 25% lipids on a dry weight basis with 80% of the complex lipid present as monogalactosyldiglycerides and digalactosyldiglycerides. The fatty acid composition of leaf lipids is dominated by the occurrence of a high proportion of unsaturated fatty acids, especially linoleic (18:2) and linolenic (18:3), along with smaller amounts of oleic (18: 1) acid. The monogalactosyldiglycerides from al-
Issue 6
~
Critical Reviews in Microbiology Downloaded from informahealthcare.com by University of California San Francisco on 12/10/14 For personal use only.
Microbiology falfa lipids contained 95% 18:3 fatty acid on a DM ba~is.'~.'~.'~ Although the forage lipid intake of ruminants is largely glycoand phospholipid, it is common practice to supplement diets with concentrates that consist largely of cereal grains or to feed high-energy, concentrate diets to frnish cattle in feedlots. The overall effect is to increase the intake of unesterified fatty acids and particularly triglycerides containing these fatty acids. The fatty acids of many seed oils normally contain high proportions of 18:2 fatty acids. Although the composition of the total fatty acids present in concentrates varies considerably it normally ranges: 16:0, 15 to 20%; 18:0, 1 to 5%; 18:1, 25 to 35%; and 18:2, 30 to O%."
'
IV. LIPID COMPOSITION OF ANAEROBIC MICROORGANISMS The microbial population of the rumen and other anaerobic ecosystems includes bacteria, ciliate protozoa, flagellate protozoa, phycomycete fungi, and a m ~ e b a s . ~ - ~The ~ ' ~ .nature '" of the lipids present in the cells is of great importance to membrane function and thus plays a vital role in determining their relative biosynthetic, enzymic, and functional aspects. In the ruminant animal, bacterial and protozoal lipids together account for 10 to 20% of the total lipid present in digesta and are of considerable importance to the postruminal supply of lipid^.'^.'^ In this section of the review, differences in lipid composition and structure of anaerobic prokaryotes (Gram-positive and Gram-negative eubacteria), methanogenic bacteria (representing archaebacteria), and anaerobic eukaryotes (ciliate protozoa and anaerobic rumen fungi) are considered. The reader is referred to comprehensive reviews on lipid structure that can only be summarized briefly here.13*'4.40"17 It should be noted that the chemical composition of bacteria is markedly influenced by environmental conditions such as substrate, temperature, growth rate, and growth factors."* Thus, lipid composition, both quantitatively and qualitatively, varies widely and is meaningful only for the experimental conditions under which it is derived. In general, prokaryotic cells are characterized by the absence of a nuclear membrane and discrete subcellular particles, whereas eukaryotic cells possess membrane bound nuclei and subcellular organelles, and differences between the lipid composition of these two groups or organisms reflects differences in their level of organization. It is also worth noting that all the identified membrane lipids of the archaebacteria are characterized by unusual structural features which are considered to be specific and therefore a useful taxonomic marker for these bacteria.4s47 Unlike eubacterial and eukaryotic lipids, which are based on ester linkages formed by condensation of alcohols and fatty acids, archaebacterial lipids are mainly isopranyl glycerol ethers. These molecules are formed by the condensation of glycerol (or more complex polyols) with isoprenoid alcohols of 20, 25, or 40 carbon at-
1991
oms.4547In addition, the C-2 configuration of glycerol in archaebacterial lipids is opposite to the conventional diglyceride configuration. These unusual structural features suggest important differences in membrane topology and raises questions related to membrane structure and function and the biochemical diversity required for life under different conditions. A. Lipid Composition of Bacteria Lipids typically represent between 3 and 20% of the cell dry weight of many microorganisms.'" The lipids present in Grampositive bacteria are limited to the cytoplasmic membranes and their invaginations, the mesosomes, and account for 4 to 7% of the dry weight of cells. The discovery of the C,, cyclopropane acid, still known as lactobacillic acid, in various Lactobacillus species stimulated the study of microbial lipids.@Grampositive species have a high proportion of branched-chain (isoand/or anteiso-) fatty acids which are less common in Gramnegative bacteria, although this generalization is not apparent among the anaerobes (Tables 2 and 3). The phospholipid composition of asporogenous Gram-positive bacteria usually contains phosphatidylglycerol and its various derivatives, including aminoacyl esters. Phosphatidylethanolamine and its derivatives are less common in Gram-positive than Gram-negative bacteria. In contrast, plasmalogens are common in anaerobes of both groups and were first described in the ruminal anaerobe Ruminococcusflavefaciens. This was also shown to apply to a variety of species of rumen bacteria.14.62*66*138 Apart from phospholipids, glycolipids (mainly glycosyldiacylglycerols) also give rise to some unusual phosphoglycolipids and constitute the hydrophobic moiety of lipoteichoic acids (Table 4). Grampositive bacteria typically contain menaquinones. Thus, Grampositive bacteria contain a variety of lipids in the cytoplasmic membrane that are involved in membrane integrity, function, and transport. The undying ultrastructural feature of Gram-negative bacteria is their complex cell envelope, consisting of an inner (cytoplasmic) membrane and an outer membrane with an intervening layer of peptidoglycan. Free (readily extractable) lipid typically accounts for 5 to 10% of the dry cell mass.41In some cases, additional lipid may come from layers external to the outer membrane. Other external layers (slime, capsule, or glycocalyx) are predominantly carbohydrate, but the presence of bound lipid has been documented in Escherichiu coli. Inclusion bodies and intracytoplasmic membranes can make a more substantial contribution to total cellular lipid. Poly-phydroxyalkanoates (PHA) are widely distributed among aerobic bacteria and under conditions of carbon and energy excess can constitute as much as 90% of cell mass. The cell envelopes of Gram-negative bacteria have a number of relevant features that concern lipid content, namely: (1) the insertion of lipid A of the lipopolysaccharidespecifically into the outer leaflet leaving the O-antigenic polysaccharide projecting out from the cell surface; (2) the presence of free or covalently attached lipo45 1
Critical Reviews In Table 2 Fatty Acid Composition Expressed as Total Fatty Acids in Whole Cells or Readily Extractable Lipids of Gram-Positive Facultative and Anaerobic Bacteria
Critical Reviews in Microbiology Downloaded from informahealthcare.com by University of California San Francisco on 12/10/14 For personal use only.
Straightchain, C-even
Organism
12:O
14:O
14:l
16:O
16:l
180
l8:l
Others
Straightchain C-odd
Enterococcus fmcalis Enterococcus faecium Luctococcus lactis Luctococcus plantarum Streptococcus cecorum Streptococcus salivarius Peptococcus asaccharolylicus Peptostreptococcus anaerobius
0
5
TP
39
6
8
32
0
Tr
0
9
49
0
5
Tr
35
5
6
34
0
2
0
12
49
0 1
8 6
1
43 41
3 5
2 4
15
16
0 0
0 0
0 0
28
Tr
27
50 50
0
2
0
34
4
13
44
3
0
0
Tr
51
Tr
3
Tr
29
6
15
28
19
0
0
0
52
+b
6
-
25
11
6
31
+
0
53
0
54
0
53
Peptostreptococcus productus Rwinococcus &us Rurninococcus flavefaciens Butyrivibrio jibrisolvens Butyrivibrio strains Group 1 Group Il Clostridium acetobutylicum
26
Lipid Metabolism in Anaerobic Ecosystems Roderick I. Mackie, Ph.D., Bryan A. White, Ph.D., and Marvin P. Bryant, Ph.D.
Critical Reviews in Microbiology Downloaded from informahealthcare.com by University of California San Francisco on 12/10/14 For personal use only.
ABSTRACT In anaerobic ecosystems, acyl lipids are initially hydrolyzed by microbial lipases with the release of free fatty acids. Glycerol, galactose, choline, and other non-fatty acid components released during hydrolysis are fermented to volatile fatty acids by the fennentative bacteria. Fatty acids are not degraded further in the rumen or other parts of the digestive tract but are subjected to extensive biohydrogenation especially in the rumen. However, in environments such as sediments and waste digestors, which have long retention times, both long and short chain fatty acids are P-oxidized to acetate by a special group of bacteria, the H,-producing syntrophs. Long chain fatty acids can also be degraded by a-oxidation. Biotransformation of bile acids, cholesterol, and steroids by intestinal microorganisms is extensive. Many rumen bacteria have specific growth requirements for fatty acids such as n-valeric, iso-valeric, 2methylbutyric, and iso-butyric acids. Some species have requirements for C,, to C,, straight-chain saturated or monoenoic fatty acids for growth.
1. OVERVIEW Unlike proteins, carbohydrates, and nucleic acids, the term “lipid” covers an extremely diverse range o hemical or molecular species. The definition of lipids is th refore simple they are classed as being sparingly soluble in water but readily soluble in organic solvents such as chloroform, hydrocarbons, alcohols, ethers, and esters. Common lipids can be loosely divided into two categories according to structure. First, those structures based on longchain fatty acids or their immediate derivatives Gcluding the natural fats and oils present in plants and animals as well as phospholipids and glycolipids which have a ubiquitous distribution.’ The second category of terpenoid lipids is based on the isoprene unit and range from the simplest
7
H 2
monoterpenes to the more complex steroid and carotenoid molecules.* The terpenoids are related and characterized by their biosynthesis, in whole or in part, from rnevalonic acid. A particularly useful system of classifying lipids is the division into “neutral” or “apolar” lipids, and “polar” or “am’ phiphilic” lipids. Thus, neutral lipids w ill include simple hydrocarbons, carotenes, triacylglycerols, wax esters, sterol esters, as well as other lipids such as fatty acids, polyprenols, and sterols in which the hydrophilic function has little impact on the overall molecular characteristics. Polar lipids include phospholipids, glycolipids, sulfolipids, some sphingolipids, oxygenated carotenoids, and chlorophylls. The nomenclature of lipids recommended by IUPAC-IUB has appeared in several journals and is listed by Ratledge and Wilkin~on.~ The great diversity in lipid structure is related to their diversity in function. Lipids can act as storage materials in animal, plant, and microbial cells, where the lipids occur typically as triacylglycerols in eukaryotes and as poly-p-hydroxyalkanoate(s) in some prokaryotes. They are responsible for the structure of cell membranes occuning mainly as phospholipids. They are associated with the photosynthetic process in plants and microorganisms not only as chlorophyll, but also as quinones and pigments involved in converting light energy into chemical energy. Lipoquinones are associated with energygenerating processes. Besides their universal role in membrane structure and function, lipids also participate in the organization of bacterial cell envelopes. They occur as lipoteichoic acids associated with the cytoplasmic membrane of Gram-positive bacteria and as lipopolysaccharidesand lipoproteins in the outer membrane of Gram-negative bacteria. Extracellular lipids are also produced by both prokaryotes and eukaryotes. These may be accompanied by extracellular enzymes such as lipases, esterases, and phospholipases which aid the growth of microorganisms on external sources of fats or hydrocarbons. The pathways of lipid biosynthesis in microorganisms are similar to those in higher organisms, although the anaerobic route of unsaturated fatty acid biosynthesis is found only in b a ~ t e r i a . ~
II. INTRODUCTION The input of lipids in anaerobic environments can be substantial especially in domestic sewage and in sewage sludge. Although lipids are quantitatively important as substrates for
Roderick I. Mackie, Ph.D. (correspondingauthor), Bryan A. White, Ph.D., and Marvin P. Bryant, Ph.D., Department of Animal Sciences 458 Animal Science Lab, University of Illinois at Urbana-Champaign, 1207 W. Gregory Drive, Urbana, IL 61801.
H
Isoprene (methylbuta-1,3-diene)
1991
449
Critical Reviews in Microbiology Downloaded from informahealthcare.com by University of California San Francisco on 12/10/14 For personal use only.
Critical Reviews In anaerobic degradation in methanogenic ecosystems they have not been studied as extensively as the degradation of carbohydrates and proteins. Many different kinds of organisms are involved in the complex process of lipid degradation and in this review the role of bacteria in this process is emphasized, although protozoa and fungi may also be i n ~ o l v e d . In ~ - gen~ eral, lipids are hydrolyzed by fermentative bacteria with the release of long- and medium-chain length free fatty acids with the glycerol and galactose moieties being fermented to volatile fatty acids, CO, and H,.5*7Fermentative bacteria are unable to oxidize the free fatty acids and this step is carried out by specialized groups of bacteria such as the H,-producing synt r o p h ~ . ~ .or ~ .some ' ~ of the acetogenic, sulfate-reducing bacteria. In the terminal stage of complete anaerobic degradation, once inorganic electron acceptors such as nitrate and sulfate are exhausted, methanogenic bacteria utilize H,, CO,, and acetate in the production of final products, CH, and C0,.*.9J2 Much of the research on anaerobic lipid degradation has focused on a single ecosystem, the rumen. In the rumen, dietary acyl lipids are first subjected to hydrolysis by microbial li~ases.".'~Free fatty acids liberated are then subjected to extensive biohydrogenation by the nunen bacteria, resulting in an enrichment of saturated fatty acids at the expense of unsaturated fatty acids. l3 Unlike sludge or anaerobic digestion in which degradation of lipids accounts for a major portion of the acetate formed, fatty acids are not degraded further in the rumen. In fact, acetate, propionate and butyrate produced during carbohydrate fermentation accumulate and are absorbed from the digestive tract and utilized as major energy sources by the ruminant animal, the mammalian partner in this mutually beneficial association. Because of the relatively short retention time of digesta in the digestive tract of the animal, the H,producing acetogens that catabolize fatty acids and the acetateutilizing methanogenic bacteria are unable to grow rapidly enough to be maintained in the system5e9except under unusual c i r c ~ m ~ t a n c eDe ~ .novo ' ~ ~ ~synthesis ~ of microbial lipids also occurs in the rumen and free acids, both saturated and unsaturated, can be incorporated into microbial cell material. As a result of these microbial transformations, rumen lipids and postruminal digesta are enriched in stearic acid (18:O) at the expense of dietary linoleic (18:2) and linolenic (18:3) a c i d ~ . ' ~ . ' ~ In this review, research on the microbiology and biochemistry of lipid degradation and synthesis in the rumen and other anaerobic ecosystems is summarized with the emphasis on research over the past 5 to 10 years. Several excellent reviews provide extensive background information and for more details of earlier work should be c o n s ~ l t e d . ~ - ~ * ' ~ * ' ~ * ' '
111. LIPID CONTENT AND COMPOSITION OF SUBSTRATES The lipid content of substrates utilized in different anaerobic ecosystems for methanogenic fermentations varies widely (Ta-
450
Volume 1
ble 1). Waste from intensively reared farm animals is often collected and used as substrate. Pig manure has the highest lipid content (8 to 19%) of the animal wastes. Domestic sewage and sewage sludge have relatively large amounts of lipids and contain a high proportion of fats, oils, and greases. Wastes from meat packing and slaughterhouses are especially high in lipids, with flotation foams containing 60 to 65% lipid on a dry basis. Generally, lipids are 75 to 95% degraded during sludge digestion and account for approximately the same amount of methane formation (20 to 30%) as the carbohydrates.L9*37*38
Table 1 Lipid Content of Substrates for Anaerobic Digestion and Dietary Lipids Consumed by Ruminants Source Cattle waste Pig waste Poultry waste Domestic refuse Domestic sewage Sewage sludge Activated sewage sludge Brewery activated sludge Abattoir activated sludge Flotation foams Slaughterhouse wastes Alfalfa, fresh Clover, red Orchard grass, fresh Ryegrass, fresh Barley, grain Brewers grains Distillers grains Corn, grain Cotton seeds Oats, grain Peanut meal soybean seeds Sunflower seed meal Wheat, grain
Lipid content (as 9% of VS or DM) 3.w.5
I . 1-19,2 1.5-2.1 6.2 10-35 8-25 5-12 10-15 15-25 60-65 25-55 2.6-3.1 2.9-5.0 3.u.9 3.2 2.1 6.5 9.8-10.3 2.24.2 23.1 5.4-6.9 6.3 18.8 8.1 1.8-2.2
Ref. 18-21 22-25 26, 27 28, 29 30-32 31, 33 34 34 34 35 35 36 36 36 36 36 36 36 36 36 36 36 36 36 36
Ruminants are herbivores and the lipids derived from forages are predominantly of leaf origin. Lipids comprise 2 to 3% of the DM of fresh forages and about 6 to 8% of the dry weight of leaf tissue. Glycolipids and phospholipids comprise approximately 95% of total leaf lipids with glycolipids comprising 70 to 80% of the complex lipids present irrespective of plant species. Most of these lipids are present in the chloroplasts of leaf tissue. Chloroplasts contain 22 to 25% lipids on a dry weight basis with 80% of the complex lipid present as monogalactosyldiglycerides and digalactosyldiglycerides. The fatty acid composition of leaf lipids is dominated by the occurrence of a high proportion of unsaturated fatty acids, especially linoleic (18:2) and linolenic (18:3), along with smaller amounts of oleic (18: 1) acid. The monogalactosyldiglycerides from al-
Issue 6
~
Critical Reviews in Microbiology Downloaded from informahealthcare.com by University of California San Francisco on 12/10/14 For personal use only.
Microbiology falfa lipids contained 95% 18:3 fatty acid on a DM ba~is.'~.'~.'~ Although the forage lipid intake of ruminants is largely glycoand phospholipid, it is common practice to supplement diets with concentrates that consist largely of cereal grains or to feed high-energy, concentrate diets to frnish cattle in feedlots. The overall effect is to increase the intake of unesterified fatty acids and particularly triglycerides containing these fatty acids. The fatty acids of many seed oils normally contain high proportions of 18:2 fatty acids. Although the composition of the total fatty acids present in concentrates varies considerably it normally ranges: 16:0, 15 to 20%; 18:0, 1 to 5%; 18:1, 25 to 35%; and 18:2, 30 to O%."
'
IV. LIPID COMPOSITION OF ANAEROBIC MICROORGANISMS The microbial population of the rumen and other anaerobic ecosystems includes bacteria, ciliate protozoa, flagellate protozoa, phycomycete fungi, and a m ~ e b a s . ~ - ~The ~ ' ~ .nature '" of the lipids present in the cells is of great importance to membrane function and thus plays a vital role in determining their relative biosynthetic, enzymic, and functional aspects. In the ruminant animal, bacterial and protozoal lipids together account for 10 to 20% of the total lipid present in digesta and are of considerable importance to the postruminal supply of lipid^.'^.'^ In this section of the review, differences in lipid composition and structure of anaerobic prokaryotes (Gram-positive and Gram-negative eubacteria), methanogenic bacteria (representing archaebacteria), and anaerobic eukaryotes (ciliate protozoa and anaerobic rumen fungi) are considered. The reader is referred to comprehensive reviews on lipid structure that can only be summarized briefly here.13*'4.40"17 It should be noted that the chemical composition of bacteria is markedly influenced by environmental conditions such as substrate, temperature, growth rate, and growth factors."* Thus, lipid composition, both quantitatively and qualitatively, varies widely and is meaningful only for the experimental conditions under which it is derived. In general, prokaryotic cells are characterized by the absence of a nuclear membrane and discrete subcellular particles, whereas eukaryotic cells possess membrane bound nuclei and subcellular organelles, and differences between the lipid composition of these two groups or organisms reflects differences in their level of organization. It is also worth noting that all the identified membrane lipids of the archaebacteria are characterized by unusual structural features which are considered to be specific and therefore a useful taxonomic marker for these bacteria.4s47 Unlike eubacterial and eukaryotic lipids, which are based on ester linkages formed by condensation of alcohols and fatty acids, archaebacterial lipids are mainly isopranyl glycerol ethers. These molecules are formed by the condensation of glycerol (or more complex polyols) with isoprenoid alcohols of 20, 25, or 40 carbon at-
1991
oms.4547In addition, the C-2 configuration of glycerol in archaebacterial lipids is opposite to the conventional diglyceride configuration. These unusual structural features suggest important differences in membrane topology and raises questions related to membrane structure and function and the biochemical diversity required for life under different conditions. A. Lipid Composition of Bacteria Lipids typically represent between 3 and 20% of the cell dry weight of many microorganisms.'" The lipids present in Grampositive bacteria are limited to the cytoplasmic membranes and their invaginations, the mesosomes, and account for 4 to 7% of the dry weight of cells. The discovery of the C,, cyclopropane acid, still known as lactobacillic acid, in various Lactobacillus species stimulated the study of microbial lipids.@Grampositive species have a high proportion of branched-chain (isoand/or anteiso-) fatty acids which are less common in Gramnegative bacteria, although this generalization is not apparent among the anaerobes (Tables 2 and 3). The phospholipid composition of asporogenous Gram-positive bacteria usually contains phosphatidylglycerol and its various derivatives, including aminoacyl esters. Phosphatidylethanolamine and its derivatives are less common in Gram-positive than Gram-negative bacteria. In contrast, plasmalogens are common in anaerobes of both groups and were first described in the ruminal anaerobe Ruminococcusflavefaciens. This was also shown to apply to a variety of species of rumen bacteria.14.62*66*138 Apart from phospholipids, glycolipids (mainly glycosyldiacylglycerols) also give rise to some unusual phosphoglycolipids and constitute the hydrophobic moiety of lipoteichoic acids (Table 4). Grampositive bacteria typically contain menaquinones. Thus, Grampositive bacteria contain a variety of lipids in the cytoplasmic membrane that are involved in membrane integrity, function, and transport. The undying ultrastructural feature of Gram-negative bacteria is their complex cell envelope, consisting of an inner (cytoplasmic) membrane and an outer membrane with an intervening layer of peptidoglycan. Free (readily extractable) lipid typically accounts for 5 to 10% of the dry cell mass.41In some cases, additional lipid may come from layers external to the outer membrane. Other external layers (slime, capsule, or glycocalyx) are predominantly carbohydrate, but the presence of bound lipid has been documented in Escherichiu coli. Inclusion bodies and intracytoplasmic membranes can make a more substantial contribution to total cellular lipid. Poly-phydroxyalkanoates (PHA) are widely distributed among aerobic bacteria and under conditions of carbon and energy excess can constitute as much as 90% of cell mass. The cell envelopes of Gram-negative bacteria have a number of relevant features that concern lipid content, namely: (1) the insertion of lipid A of the lipopolysaccharidespecifically into the outer leaflet leaving the O-antigenic polysaccharide projecting out from the cell surface; (2) the presence of free or covalently attached lipo45 1
Critical Reviews In Table 2 Fatty Acid Composition Expressed as Total Fatty Acids in Whole Cells or Readily Extractable Lipids of Gram-Positive Facultative and Anaerobic Bacteria
Critical Reviews in Microbiology Downloaded from informahealthcare.com by University of California San Francisco on 12/10/14 For personal use only.
Straightchain, C-even
Organism
12:O
14:O
14:l
16:O
16:l
180
l8:l
Others
Straightchain C-odd
Enterococcus fmcalis Enterococcus faecium Luctococcus lactis Luctococcus plantarum Streptococcus cecorum Streptococcus salivarius Peptococcus asaccharolylicus Peptostreptococcus anaerobius
0
5
TP
39
6
8
32
0
Tr
0
9
49
0
5
Tr
35
5
6
34
0
2
0
12
49
0 1
8 6
1
43 41
3 5
2 4
15
16
0 0
0 0
0 0
28
Tr
27
50 50
0
2
0
34
4
13
44
3
0
0
Tr
51
Tr
3
Tr
29
6
15
28
19
0
0
0
52
+b
6
-
25
11
6
31
+
0
53
0
54
0
53
Peptostreptococcus productus Rwinococcus &us Rurninococcus flavefaciens Butyrivibrio jibrisolvens Butyrivibrio strains Group 1 Group Il Clostridium acetobutylicum
26
