34V.IvanouaFactor(s) of medium+Biotic element 2Biotic element 1?+十Factor(s) ofmediumbFactor(s) ofmedium0Biotic element 2Biotic element 100Factor(s) of mediumcFactor(s)ofmediumOBiotic element 2Biotic element 1+Factor(s) of mediumFig.2.3.Examples of mutual microbial interactions: (a),both microbial groups are exchanging withgrowth factors or nutrients; (b), both microbial groups diminish negative factors of medium; (c),removal of oxygen bygroup 2 creates anaerobic conditionsfor fixation of nitrogenbygroup1 whichsuppliesnitrogen compoundsforgroup2.Mutual interactions are facilitated by close physical proximityin microcolonies,biofilmsand flocs.Physiological cooperation in biofilm or aggregates is supplemented and supportedby its spatial structure, i.e., formation of microhabitats for individual populations. Someexamplesofmutualismareasfollows:1.Syntrophy ("co-eating"), both microbial groups supply growth factors or nutrients.2.Sequential biodegradation of xenobiotics when the product of biodegradation inhibits biodegra-dation
34 V. Ivanov b Factor(s) of medium Biotic element 1 Biotic element 2 Factor(s) of medium a Factor(s) of medium Biotic element 1 Biotic element 2 Factor(s) of medium Factor(s) of medium Biotic element 1 Biotic element 2 Factor(s) of medium c Fig. 2.3. Examples of mutual microbial interactions: (a), both microbial groups are exchanging with growth factors or nutrients; (b), both microbial groups diminish negative factors of medium; (c), removal of oxygen by group 2 creates anaerobic conditions for fixation of nitrogen by group 1 which supplies nitrogen compounds for group 2. Mutual interactions are facilitated by close physical proximity in microcolonies, biofilms and flocs. Physiological cooperation in biofilm or aggregates is supplemented and supported by its spatial structure, i.e., formation of microhabitats for individual populations. Some examples of mutualism are as follows: 1. Syntrophy (“co-eating”), both microbial groups supply growth factors or nutrients. 2. Sequential biodegradation of xenobiotics when the product of biodegradation inhibits biodegradation
35MicrobiologyofEnvironmentalEngineeringSystems3.Cyclingof elementby two microbial groups:(4)Phototrophicbacteria+light+H2S+CO2→S+organics(5)Facultativeanaerobicbacteria+S+organics-→H2S+CO24.Removal ofoxygen by heterotrophic bacteria creates anaerobic conditions for fixation ofnitrogenbyphototrophic cyanobacteria, which supply nitrogen compoundsforheterotrophic bacteria.Positive interactions between animals and microorganisms are common and often essentialfor animals.Microorganisms can improve the digestion and assimilation of food by ani-mals;produce growth factors,like vitamins and essential aminoacids foranimals andkeepout thepathogenic microorganismsfrom the surfaceand cavities of macroorganisms.Forexample, ants and termites cultivate cellulose-degrading fungi in their chambers to enhancethe feeding value of plant material. Insects provide cellulose-degrading fungi with favorableconditions, and a supply of cellulose and mineral components. Another example is interactionsbetween microorganisms and human organisms.Thehumanbody containscomplex and stablemicrobial communities on the skin, hairs, body cavities, and within the gastrointestinal tract.Macroorganisms providefavorable conditions and supplynutrients tomicroorganisms, whichproduce some vitamins andkeep out the pathogenicmicroorganisms from the skin and surfaceofcavities.Symbiotic mutualism, or simply symbiosis, means that two groups cannot live separately.This is commonly the case in the interaction between macroorganisms and microorganismsManyprotozoa have symbiotic relations with bacteria and algae, often including them intothecell as endosymbionts.Bacterial endosymbionts supply the growthfactors to the protozoanpartner.A well-known example is the symbiosis of ruminant animals (cow, deer, sheep)and anaerobic, cellulose-degrading microorganisms in their rumen.Ruminant animals ensurecrushed organics andmineral components,optimal pH and temperature,andmicroorganismshydrolyze and transform cellulose to assimilated fatty acids.Positive interactions between plants and microorganisms are common and often essentialfor plants. Epiphytic microorganisms live on aerial plant structures such as stems, leaves andfruits.The habitat and microorganisms on the plant leaves is called phyllosphere.The yeastsand lactic acid bacteria, for example, dominate in the phyllosphere. They receive carbohy-drates and vitamins from the plant. High microbial activity occurs also in the soil surroundingthe roots,called the rhizosphere.Organic compounds that stimulateheterotrophicmicrobesare excreted through the roots. Some fungi are integrated into the roots and contribute to plantmineral nutrition.This type of symbiotic interaction is called mycorrhizae.An example ofmycorrhizae is the interaction between pine and fungi. Fungi integrated into the roots of pinecontribute to plant mineral nutrition in exchange for a supply of organic nutrition from theplant.Symbiotic mutualism of plants and microorganisms is a common interaction.A well-knownexample is the symbiotic fixation of atmospheric nitrogen, which is a major reservoir ofnitrogen for life. The roots of some plants are invaded by nitrogen-fixing bacteria, mainlyfrom the genus Rhizobium, which form tumor-like aggregates (nodule), where the bacteriaare transformed into large cells (bacteroids) capable of fixing N2 from air.A plant supplies
Microbiology of Environmental Engineering Systems 35 3. Cycling of element by two microbial groups: Phototrophic bacteria + light + H2S + CO2 → S + organics (4) Facultative anaerobic bacteria + S + organics → H2S + CO2 (5) 4. Removal of oxygen by heterotrophic bacteria creates anaerobic conditions for fixation of nitrogen by phototrophic cyanobacteria, which supply nitrogen compounds for heterotrophic bacteria. Positive interactions between animals and microorganisms are common and often essential for animals. Microorganisms can improve the digestion and assimilation of food by animals; produce growth factors, like vitamins and essential aminoacids for animals and keep out the pathogenic microorganisms from the surface and cavities of macroorganisms. For example, ants and termites cultivate cellulose-degrading fungi in their chambers to enhance the feeding value of plant material. Insects provide cellulose-degrading fungi with favorable conditions, and a supply of cellulose and mineral components. Another example is interactions between microorganisms and human organisms. The human body contains complex and stable microbial communities on the skin, hairs, body cavities, and within the gastrointestinal tract. Macroorganisms provide favorable conditions and supply nutrients to microorganisms, which produce some vitamins and keep out the pathogenic microorganisms from the skin and surface of cavities. Symbiotic mutualism, or simply symbiosis, means that two groups cannot live separately. This is commonly the case in the interaction between macroorganisms and microorganisms. Many protozoa have symbiotic relations with bacteria and algae, often including them into the cell as endosymbionts. Bacterial endosymbionts supply the growth factors to the protozoan partner. A well-known example is the symbiosis of ruminant animals (cow, deer, sheep) and anaerobic, cellulose-degrading microorganisms in their rumen. Ruminant animals ensure crushed organics and mineral components, optimal pH and temperature, and microorganisms hydrolyze and transform cellulose to assimilated fatty acids. Positive interactions between plants and microorganisms are common and often essential for plants. Epiphytic microorganisms live on aerial plant structures such as stems, leaves and fruits. The habitat and microorganisms on the plant leaves is called phyllosphere. The yeasts and lactic acid bacteria, for example, dominate in the phyllosphere. They receive carbohydrates and vitamins from the plant. High microbial activity occurs also in the soil surrounding the roots, called the rhizosphere. Organic compounds that stimulate heterotrophic microbes are excreted through the roots. Some fungi are integrated into the roots and contribute to plant mineral nutrition. This type of symbiotic interaction is called mycorrhizae. An example of mycorrhizae is the interaction between pine and fungi. Fungi integrated into the roots of pine contribute to plant mineral nutrition in exchange for a supply of organic nutrition from the plant. Symbiotic mutualism of plants and microorganisms is a common interaction. A well-known example is the symbiotic fixation of atmospheric nitrogen, which is a major reservoir of nitrogen for life. The roots of some plants are invaded by nitrogen-fixing bacteria, mainly from the genus Rhizobium, which form tumor-like aggregates (nodule), where the bacteria are transformed into large cells (bacteroids) capable of fixing N2 from air. A plant supplies
36V.Ivanovthe bacteroids with organic and mineral feed and the bacteroids supply organic nitrogen tothe plant. Symbiotic relations ensure the existence of lichens where photosynthetic algae orcyanobacterial component of lichens produce organic matter and microscopic fungi providemineral nutrient transport and the mechanical framefor the photosynthetic organisms.Mostlichens are resistant to extremetemperatures and drying and are capable of fixing nitrogen andoccupying hostile environments.Neutral competitionbetween thebiotic elements(organisms/populations/groups)meanscompetition by the rate of nutrients consumption or growth rate.Theremay also be neutralcompetition by affinity with the nutrients orby resistance to environmental factors unfavor-able for growth.It is the most typical interaction between aquatic natural ecosystems andwastewatertreatmentengineeringsystems.Amensalism is an active competition in which onebiotic elementproduces a substancethat inhibits the growth of another biotic element. There may be, for example, changes inpH caused by the production of inorganic and organic acids by one population. Neutral com-petition and amensalism are the main mechanisms for forming an enrichment culture whereone or some species dominate after cultivation of an environmental sample.The productionof antibioticsis a specific application of amensalism becauseantibiotic is a substanceableto, at low concentrations,negatively affect the growth of sensitive cells.Antibiotic-producingmicroorganisms dominate in rich environments with optimal conditions for growth, i.e., in thebiotops where neutral competition is not sufficient to ensuredomination of one biotic element.These biotops are soil,phyllosphere, skin or cavities of animals, but not aquatic biotops witha low concentration of nutrients.Antagonism is the active competition between two biotic elements, ie., competitionenhanced byspecific tools suchasexcretion of chemical substances,includingantibioticsby two competing biotic elements.Predation occurs when one organism engulfs and digests another organism.A typicalpredator-prey relationship exists between predator protozoa and bacteria.Therefore,thepredator protozoa improve the bacteriological quality of the effluent after aerobic wastewatertreatment because it helps to reduce the number offree-living bacteria.Parasitism is a very common interaction between microorganisms and macroorganismsand between different microorganisms.The benefiting parasite derives its nutritional require-ments from the host, which is the harmed organism. All viruses are parasites of bacteria.fungi, algae, plants and animals. Some prokaryotes are parasites of prokaryotes.For example,Bdellovibrio spp., small curved cells, are parasites of Gram-negative bacteria, and Vampiro-coccus spp.sucks the cytoplasm out of anotherbacterium.Enumeration of microbial parasitesin the environmental sample by the zones of lysis on a Petri dish with a layer of specificbacteria is the simplest nondirect way to evaluate the pollution of environment with thesebacteria.Growthofbacterial viruses(bacteriophages)can deteriorate the industrial cultivationprocess because of spontaneous lyses of bacterial cells.Bacteriophages are widely used ingenetic engineering of bacterial strains as the vectorfor transferof defined genes into bacterialcells.Plant parasites are represented by phytopathogenic viruses, prokaryotes and fungi. Theseparasites cause plant diseases. Typical stages of disease are as follows:
36 V. Ivanov the bacteroids with organic and mineral feed and the bacteroids supply organic nitrogen to the plant. Symbiotic relations ensure the existence of lichens where photosynthetic algae or cyanobacterial component of lichens produce organic matter and microscopic fungi provide mineral nutrient transport and the mechanical frame for the photosynthetic organisms. Most lichens are resistant to extreme temperatures and drying and are capable of fixing nitrogen and occupying hostile environments. Neutral competition between the biotic elements (organisms/populations/groups) means competition by the rate of nutrients consumption or growth rate. There may also be neutral competition by affinity with the nutrients or by resistance to environmental factors unfavorable for growth. It is the most typical interaction between aquatic natural ecosystems and wastewater treatment engineering systems. Amensalism is an active competition in which one biotic element produces a substance that inhibits the growth of another biotic element. There may be, for example, changes in pH caused by the production of inorganic and organic acids by one population. Neutral competition and amensalism are the main mechanisms for forming an enrichment culture where one or some species dominate after cultivation of an environmental sample. The production of antibiotics is a specific application of amensalism because antibiotic is a substance able to, at low concentrations, negatively affect the growth of sensitive cells. Antibiotic-producing microorganisms dominate in rich environments with optimal conditions for growth, i.e., in the biotops where neutral competition is not sufficient to ensure domination of one biotic element. These biotops are soil, phyllosphere, skin or cavities of animals, but not aquatic biotops with a low concentration of nutrients. Antagonism is the active competition between two biotic elements, i.e., competition enhanced by specific tools such as excretion of chemical substances, including antibiotics, by two competing biotic elements. Predation occurs when one organism engulfs and digests another organism. A typical predator–prey relationship exists between predator protozoa and bacteria. Therefore, the predator protozoa improve the bacteriological quality of the effluent after aerobic wastewater treatment because it helps to reduce the number of free-living bacteria. Parasitism is a very common interaction between microorganisms and macroorganisms, and between different microorganisms. The benefiting parasite derives its nutritional requirements from the host, which is the harmed organism. All viruses are parasites of bacteria, fungi, algae, plants and animals. Some prokaryotes are parasites of prokaryotes. For example, Bdellovibrio spp., small curved cells, are parasites of Gram-negative bacteria, and Vampirococcus spp. sucks the cytoplasm out of another bacterium. Enumeration of microbial parasites in the environmental sample by the zones of lysis on a Petri dish with a layer of specific bacteria is the simplest nondirect way to evaluate the pollution of environment with these bacteria. Growth of bacterial viruses (bacteriophages) can deteriorate the industrial cultivation process because of spontaneous lyses of bacterial cells. Bacteriophages are widely used in genetic engineering of bacterial strains as the vector for transfer of defined genes into bacterial cells. Plant parasites are represented by phytopathogenic viruses, prokaryotes and fungi. These parasites cause plant diseases. Typical stages of disease are as follows:
37Microbiology of Environmental Engineering Systems1.Contact of the microorganismwith the plant.2.Entry of the pathogen into the plant.3.Growthoftheinfectingmicroorganism4.Developmentofplantdiseasesymptoms.Microbial pathogens disrupt normal plant functions by producing enzymes, toxins and growthregulators. Some plant pathogens such as white-rot fungi or bacteria from genus Pseudomonascan degrade xenobiotics and are widely used in environmental engineering.Therefore, the riskofplant infection mustbeaccountedforinenvironmental biotechnologyoperations,especiallyduring soil bioremediation.Parasites of human and animals are represented bypathogenic viruses,prokaryotes, fungior protozoa.Thepathogenic (infectious)microorganisms grow in animal tissue and cancause diseases in macroorganisms. Saprophytic microorganisms feed on dead organic matter.Opportunistic pathogens arenormallyharmless but have the potential to be pathogens fordebilitated orimmunocompromised organisms.Infection refers to the disease transmission caused by the transfer of pathogenic microor-ganisms from the environment orfrom onemacroorganism to another.Infectious microor-ganisms can enter a human through direct contact between individuals or reservoir-to-personcontact. The diseases may be conventionally distinguished as air-borne, water-borne, soil-borne and food-borne infectious diseases. When infectious agents are spread by an insectsuch as a mosquito, fea, lice, biting fly or tick, they are referred to as vectors.Infectious diseases still account for 30-50% of deaths indeveloping countries becauseof poor sanitation.By comparison,mortality frominfectious diseases is 10 times smaller indeveloped countries. Transmission of water-borne diseases is directly related to the bacteri-ological quality of water and effluent of wastewater treatment plants.Sources of pathogensotherthan sewage outlets are wildlifewatersheds,farms and landfills.Theprevention ofoutbreaks of water-borne and air-borne diseases is one of the main goals of environmentalbiotechnology. Environmental engineers and epidemiologists must work closely to identifythe reason of outbreak, find its source (reservoir), define themajor means of transmission ofinfectious microorganisms, and to develop a way to stop ordiminish the scaleof outbreak.Factors of pathogenicityincludethefollowing abilities of microorganisms:1.Production of exotoxins, which are extracellular proteins. In this case, host damage can occur atsitesfar removed from alocalizedfocus of infection.Forexample,anaerobicbacteria Clostrididtetani can be introduced from the soil into the body with deep puncture wounds. If the woundbecomes anaerobic, the microorganism can grow and release its tetanus toxin, which causesspastic paralysis.2.Production of enterotoxins, which are the exotoxins that act in the small intestine.These causediarrhea, the secretion of fluid into the intestinal passage.3.Production of endotoxins, which are lipopolysaccharides of the outer membrane of Gram-negativebacteria.Endotoxinsarelesstoxicthanexotoxins.4.Formation of microstructures (fimbriae,flagellum)and macromoleculesfor specific adherenceofmicrobial cells or viruses to a host cell.5.Formation of cell structures (capsule) and macromolecules (O-antigen) protecting microbial cellsfrom thereaction of a hostmacroorganism
Microbiology of Environmental Engineering Systems 37 1. Contact of the microorganism with the plant. 2. Entry of the pathogen into the plant. 3. Growth of the infecting microorganism. 4. Development of plant disease symptoms. Microbial pathogens disrupt normal plant functions by producing enzymes, toxins and growth regulators. Some plant pathogens such as white-rot fungi or bacteria from genus Pseudomonas can degrade xenobiotics and are widely used in environmental engineering. Therefore, the risk of plant infection must be accounted for in environmental biotechnology operations, especially during soil bioremediation. Parasites of human and animals are represented by pathogenic viruses, prokaryotes, fungi or protozoa. The pathogenic (infectious) microorganisms grow in animal tissue and can cause diseases in macroorganisms. Saprophytic microorganisms feed on dead organic matter. Opportunistic pathogens are normally harmless but have the potential to be pathogens for debilitated or immunocompromised organisms. Infection refers to the disease transmission caused by the transfer of pathogenic microorganisms from the environment or from one macroorganism to another. Infectious microorganisms can enter a human through direct contact between individuals or reservoir-to-person contact. The diseases may be conventionally distinguished as air-borne, water-borne, soilborne and food-borne infectious diseases. When infectious agents are spread by an insect such as a mosquito, flea, lice, biting fly or tick, they are referred to as vectors. Infectious diseases still account for 30–50% of deaths in developing countries because of poor sanitation. By comparison, mortality from infectious diseases is 10 times smaller in developed countries. Transmission of water-borne diseases is directly related to the bacteriological quality of water and effluent of wastewater treatment plants. Sources of pathogens other than sewage outlets are wildlife watersheds, farms and landfills. The prevention of outbreaks of water-borne and air-borne diseases is one of the main goals of environmental biotechnology. Environmental engineers and epidemiologists must work closely to identify the reason of outbreak, find its source (reservoir), define the major means of transmission of infectious microorganisms, and to develop a way to stop or diminish the scale of outbreak. Factors of pathogenicity include the following abilities of microorganisms: 1. Production of exotoxins, which are extracellular proteins. In this case, host damage can occur at sites far removed from a localized focus of infection. For example, anaerobic bacteria Clostridia tetani can be introduced from the soil into the body with deep puncture wounds. If the wound becomes anaerobic, the microorganism can grow and release its tetanus toxin, which causes spastic paralysis. 2. Production of enterotoxins, which are the exotoxins that act in the small intestine. These cause diarrhea, the secretion of fluid into the intestinal passage. 3. Production of endotoxins, which are lipopolysaccharides of the outer membrane of Gramnegative bacteria. Endotoxins are less toxic than exotoxins. 4. Formation of microstructures (fimbriae, flagellum) and macromolecules for specific adherence of microbial cells or viruses to a host cell. 5. Formation of cell structures (capsule) and macromolecules (O-antigen) protecting microbial cells from the reaction of a host macroorganism
38V.IvanovWater-borne pathogens enter the host bodyby ingestion of cells,cysts or viral particles.Themost common water-borne pathogenic bacteria are pathogenic strains of Escherichiacoli,Leptospira spp., Vibrio cholera, Shigella spp., Salmonella spp., and Campylobacterspp.Two protozoans of major concern as water-borne pathogens are Giardia intestinalis,Cryptosporidium spp.and Entamoebiahistolytica.Onetoten ingested cysts of Giardia cancause diarrhea.There are over 100 known water-borne human enteric viruses.An indicator microorganism is a conventionally selected microorganism or group ofmicroorganisms used to determine the risk of water-borne infection associated with fecalcontamination of water from humans or animals.There is a great variety ofpathogenicorganisms in water, and detection of each one in order to monitor water quality is an expensiveoperation.Anindicatormicroorganismmust be ofthesame originand have similarphysio-logical properties as some group of pathogens, and can be easilydetected or enumerated inawatersample.Commonindicatorsofwaterpollutionwithenteropathogens(mainagentsofwater-borne diseases)from feces of warm-blooded animals are the numbers of the cells ofE.coli (fecal coliforms),some Streptococcus spp.(fecal streptococci)or anaerobic Clostrid-ium spp.The concentration of coliforms is usuallyless than1cell/mLin treated drinking waterand more than somemillion(1o°)cells/mL in sewage.Theconcentration of heterotrophic bac-teria in water determined by heterotrophicplate count (HPC)is also important bacteriologicalparameter of waterquality.The concentration of anaerobic bacteria from genera Clostridium.Bifidobacterium or Bacteroides may beconsidereda good indicator of fecal pollution of waterbecausetheircontent infeces insome orderslargerthanthecontent of coliforms.There areno indicator organisms for protozoan cysts and viruses becauseof the specific release andsurvivalofeverystrain.3.MICROBIALGROWTHANDDEATH3.1.NutrientsandMediaThe elemental composition of biomass can be shown approximately by the formulaCHi.gOo.sNo.2. The average content of carbon in a microbial biomass is approximately 50%.Theexactelemental composition canbedeterminedbyanautomaticCOHNanalyzerandused in the design of thebiotechnological process.Forexample,the aerobic growth ofbiomass (CHi.gOo.sNo.2)and biodegradation of carbohydrates shown by formula CH,O canbe described by thefollowing equations:(6)X <CH2O>+X,O2→X,H20+X,CO2which shows oxidation of carbohydrates togenerate energy used for growth:(7)X2 < CH,0 > +X,0.502 → X, [2H] + X,C02which shows oxidation of carbohydrates to generate reducing equivalents [2H] used forbiomass synthesis; and(8)X,CH,0+X,0.2NH3+X,0.1[2H]→X,CH1.8O0.5No.2+X,0.5H20which shows assimilation of carbon from carbohydratestobiomass
38 V. Ivanov Water-borne pathogens enter the host body by ingestion of cells, cysts or viral particles. The most common water-borne pathogenic bacteria are pathogenic strains of Escherichia coli, Leptospira spp., Vibrio cholera, Shigella spp., Salmonella spp., and Campylobacter spp. Two protozoans of major concern as water-borne pathogens are Giardia intestinalis, Cryptosporidium spp. and Entamoebia histolytica. One to ten ingested cysts of Giardia can cause diarrhea. There are over 100 known water-borne human enteric viruses. An indicator microorganism is a conventionally selected microorganism or group of microorganisms used to determine the risk of water-borne infection associated with fecal contamination of water from humans or animals. There is a great variety of pathogenic organisms in water, and detection of each one in order to monitor water quality is an expensive operation. An indicator microorganism must be of the same origin and have similar physiological properties as some group of pathogens, and can be easily detected or enumerated in a water sample. Common indicators of water pollution with enteropathogens (main agents of water-borne diseases) from feces of warm-blooded animals are the numbers of the cells of E. coli (fecal coliforms), some Streptococcus spp. (fecal streptococci) or anaerobic Clostridium spp. The concentration of coliforms is usually less than 1 cell/mL in treated drinking water and more than some million (106) cells/mL in sewage. The concentration of heterotrophic bacteria in water determined by heterotrophic plate count (HPC) is also important bacteriological parameter of water quality. The concentration of anaerobic bacteria from genera Clostridium, Bifidobacterium or Bacteroides may be considered a good indicator of fecal pollution of water because their content in feces in some orders larger than the content of coliforms. There are no indicator organisms for protozoan cysts and viruses because of the specific release and survival of every strain. 3. MICROBIAL GROWTH AND DEATH 3.1. Nutrients and Media The elemental composition of biomass can be shown approximately by the formula CH1.8O0.5N0.2. The average content of carbon in a microbial biomass is approximately 50%. The exact elemental composition can be determined by an automatic COHN analyzer and used in the design of the biotechnological process. For example, the aerobic growth of biomass (CH1.8O0.5N0.2) and biodegradation of carbohydrates shown by formula CH2O can be described by the following equations: X1 < CH2O > +X1O2 → X1H2O + X1CO2 (6) which shows oxidation of carbohydrates to generate energy used for growth; X2 < CH2O > +X20.5O2 → X2 [2H] + X2CO2 (7) which shows oxidation of carbohydrates to generate reducing equivalents [2H] used for biomass synthesis; and X3CH2O + X30.2NH3 + X30.1 [2H] → X3CH1.8O0.5N0.2 + X30.5H2O (8) which shows assimilation of carbon from carbohydrates to biomass