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Jörg Strunkheide, Dr. Ing. (Sankt Augustin, Germany)
Folic acid sources and medical application
Vitamins are essential nutritional compounds that contribute to a variety of functions, includingmetabolic maintenance and cell growth /1/. Each vitamin has particular roles that cannot befulfilled by other vitamins in the same manner. Folic acid, often cited as a "deficient" vitamin,plays an important role of which many are not aware, although it is present in many foods, suchas fruit, vegetables and whole grain products. The first scientific encounter with folic acid wasin 1931 in a study by Wills, in which eating yeast was found to correct anemia duringpregnancy, and Day, who found that moneys with aplastic anemia due to poor nutrition couldbe cured with liver extracts. In 1940, Snell et al. described an essential factor for the growth ofLactobacillus casei. Pure folic acid was isolated three years later by Stokstad and crystallized byPfiffner. The structure of this antianemia, growth or Lactobacillus casei-factor, to mention onlya few of the terms then used to refer to folic acid, was elucidated in 1946 by Angier et al. bymeans of degradation reactions and synthesis (reviewed by Koebnick /2/). Most of thefunctions performed by folic acid in the transfer of C1 units in organisms were determined by1951 /3/. After that, little attention was paid to folic acid for a longer period of time. Recently,however, attention has been focused on it once again, because the latest research shows that it isnot only a critical vitamin for nutritional maintenance, but it is crucial medical factor forembryonic development and it has a positive effect on vascular diseases, leukemia as well as onpsychological health and the brain. Folic acid does not exist in a pure state naturally. It is a"pure synthetic product obtained through isolation" /4/ and is only found it foods to which folicacid has been added as a supplement, such as some breakfast cereals, salt and flour. The name"folic acid" is derived from the Latin word "folium" (leaf), because the substance was firstisolated in 1941 from four tons of dried spinach leaves /5/.
Structure of folic acid
Folic acid belongs to the group of water-soluble B vitamins (formerly known as vitamin B9 orVitamin BC) and refers to a group of over 100 compounds with very similar properties (folates).
The term "folates" refers to the folic acid derivatives occuring naturally in plant and animalnutrition, whereas the term "folic acid" refers only to pteroylglutamic acid. Folic acid stands forfor N-[4-(2-amino-3,4-dihydro-4-oxo-6-pteridinylmethylamino)-benzoyl]-L-glutamic acid andbesteht of a 2-Amino-4-hydroxypteridine ring to which the amino group of p-aminobenzoic acidis bonded via the C6 methylene group and where the carboxyl group forms an amide bond withL-glutamic acid. The substructure formed by the pteridine ring system and p-aminobenzoic acidis referred to as pteroic acid (Figure 1).
Figure 1:
Structure of folic acid (see Kahle /1 /)
The first building block of folic acid, the pteridine ring, is the basic skeleton of the pteridinesand is found in hormones and insect dyes. The second building block of folic acid, p-aminobenzoic (PABA), is a key growth hormone for bacteria (also previously known as growthhormone H or vitamin H'). Bacteria require PABA for the synthesis of folic acid. L-glutamicacid, the final folic acid component, is an important transmitter in the central nervous system. Itis also used in the food industry as a flavor enhancer, mostly in the form of monosodiumglutamate (MSG).
More than half of the folic acid compounds exist as polyglutamates, i.e. up to eight glutaminecomponents are bound to the actual folic acid molecule via the gamma-carboyl group(pteroylpolyglutamic acids). These must first be split off during the digrestive processes,whereas the monoglutamates can be more easily absorbed in the intestine as "free" folic acid.
The bioavailability of naturally occuring folates is thus only about 50 to 70%, whereassupplemented synthetic folic has an availability of up to 95% and folic acid in pill form is nearly100% /1/.
The biologically active forms of folic acid are 5,6,7,8-tetrahydrofolic acid (THF) and itsderivates. There are four different coenzyme forms found in humans: 10-formyl-THF (alsoknown as N-10-formyltetrahydrofolate, Citrovorum factor or Leucovorin); 5,10-methyl-THF;5,10-methylene-THF and 5-methyl-THF /4/.
The role of folic acid in metabolic processes
In its active form, folic acid is involved in key metabolic processes that can be subdivided intothe categories of protein metabolism and nucleic acid metabolism. Folic acid coenzymesparticipate in the degradation and formation of various amino acids. Thus THF is involved inthe degradation of histidine as well as the conversion of serine to glycine and of homocystein tomethionine. Folic acid has an important influence on DNA synthesis and protein biosynthesisand thus a high significance for fundamental life processes /6/, so that a deficiency of folic acidis recognizable by specific dysfunctions.
The tetrahydrofolic acid 5,6,7,8-THF is the biologically active form of folic acid. Figure 2shows its formation from folic acid.
Figure 2:
Formation of tetrahydrofolic acid (see Reuter /6 /)
THF is created by the reduction of folic acid via the dihydrofolic acid (DHF) intermediatecatalyzed by the two NADPH/H+ dependent enzymes, folate reductase and dihydrofolatereductase. THF and its derivates consitute the coenzymes for transmission of C1 units such asmethyl, formyl, formate or hydroxymethyl functional groups. The carriers of these C1-units inthe "one-carbon metabolism" are always the nitrogen atoms at the fifth or tenth position of thepteroyl group. In general, the transfer reactions between the donor and acceptor take placewithout a change in oxidation state /1/.
The transfer can be divided in to three stages: b) Activation of the complex via isomerization C) Transmission of the C1-unit to the acceptor molecule.
Folic acid – from medical use to waste water technology
In the biocoenosis of sewage treatment plants as well, there is too little of the vitamin folic acid,due to its limited stability in aqeous solution and it therefore has a limiting effect on themetabolic steps in the process. The product DOSFOLAT®XS, which can be added to thetreatment plant, contains folic acid in stabilized form as its active ingredient. Here, the folic acidserves the particular function of regulating the 1-carbon metabolism, i.e. the breakdown offormic acid with oxygen to carbon dioxide and water /7/. Not only does DOSFOLAT®XScontain sufficient folic acid, but additional biochemical cofactors as well which actsynergistically to effect a fast conversion of metabolic activity in the activated sludge process.
Without folic acid, the metabolic processes in the cell membrane would occur at a significantlylower rate despite sufficient supply of air. Without folic acid, key parts of the biocoenosis workwith a multi-step, slow "primitive metabolism" - such as that which is prevalent for example inthe deep sea, where there is also no folic acid. No habituation has been observed in the process even after years of DOSFOLAT® use. DOSFOLAT®XS is available as a concentrate that iseasy to administer and has an effective delivered and has the effective power per kilogram ofabout 25 to 30 kg of unstabilized folic acid.
Systematic evaluation of operations data with the use of DOSFOLAT®XS in more than 60municipal and industrial sewage treatment plants North and outh America from 1995 to 2003provided the basis for reliable deployment criteria for DOSFOLAT®XS for various biologicalsewage treatment facilities. Here it could be proven that the influence of DOSFOLAT®XS in theactivated sludge process significantly reduced the production of excess sludge by about 50%/8/. In the meantime there are about a dozen sewage treatment plants in Germany where folicacid is being used to reduce excess sludge and increase process stability. This will be discussedin the report to follow.
Dosing station:Dosing of the stabilized folic acid takes place in the return sludge stream (Figure 3). Care shouldbe taken that there is sufficient turbulence at the dosing station to ensure thorough mixing in ofthe folic acid. The precipitant dosing station (P elimination) must not be adjacent to the folic aciddosing station.
Figure 3: Dosing system for stabilized folic acid (sewage treatment plant in Bavaria
an der Isar)
Dosing rate:The dosing rate of the stabilized folic acid is designed for moderate dry weather runoff and is tobe added continuously throughout the 24 hour day. The folic acid dosing rate required forexample for a dry weather runoff of 5,000 m_/d is determined as follows: • First 15 days at 0.5 ppm ("shock dosing"), 2.5 liters per day of DOSFOLAT®XS (concentrate), orat a dilution of 1:400 the daily dosage of DOSFOLAT®XS solution would be 1000 liters(required max. pumping rate: 1000/24 = 41.7 liters/hr).
• After that, an operating dosage of 0.1 ppm is applied, 0.5 liters per day of DOSFOLAT®XS (concentrate), or.
at a dilution of 1:400 the daily dosage of DOSFOLAT®XS solution would be 200 liters(required max. pumping rate: 200/24 = 8.3 liters/hr).
Excess sludge discharge procedure
• First 15 days at 0.5 ppm ("shock dosing“): After beginning dosage of the folic acid, the discharge routine for sludge, i.e. the dailyremoval of excess sludge (tons of solid material per day), is to be kept the same as the folic acid addition. During this period, the folic acid slowly takes effect and thebiocoenosis achieves a higher intensity in the C1-metabolic process. The self-adjustingbiocoenosis is documented by microscopic images. The acceleration of the 1-carbonmetabolic process during this period decreases the daily growth of sludge and thus increasesthe age of the sludge. Consequently, no increase of the solids content in the activated sludgetank is to be expected at the same BSB load in the feed to the sludge tank.
• After that, an operating dosage of 0.1 ppm is applied: After the 15-day "breaking-in phase", the discharge of excess sludge is reduced in stages –depending on the processing technology of the sewage treatment plant, by about 10% perweek for example. The daily sludge production will continue to decline, accompanied by afurther increase in the age of the sludge. The solid material content in the activation tank canalso be, analogous to experience with other operating materials such as tensides /9/ keptnearly constant (variations of the solid material content in the tank however can always beobserved as a consequence of rainfall events in mixed systems or changes in the supplyloads). The reduction of excess sludge discharge is to be continued in stages until anequilibrium state has established itself between the discharge and the daily production ofexcess sludge in the activation tank. This state of equilibrium has been reached if furtherreduction of the excess sludge discharge causes the solid content in the activation tank tostart increasing again; thus at this point less excess sludge is removed than is actuallyformed in the activation tank. Then it may be necessary to increase the removal by a fewpercent in order to reach the equilibrium state again. Im subsequent Zeitraum wird dann derÜberschussschlammabzug – wie er sich im equilibrium state eingestellt hat – unverändertbeibehalten. A "readjustment" of the excess sludge in the subsequent period of operation isonly necessary if the load situation at the activation stage changes.
Documenting the effects of treatment
Mass balancing /9/ has proven to be a useful tool for determining the biologically reducedincidence of excess sludge, regardless of whether the effect of the DOSFOLAT®XSdocumented in the before/after comparison is the specific biological excess sludge production or the absolute biological excess sludge masses Which procedure is applicable depends on the operating data available and the load situation ofthe biological stage (individual case observation) in the before and after comparison.
Documentation of the effect via the absolute biological excess sludge masses can be done if thesupply loads and the solids content in the activation tank (equal sludge load) are nearly the sameboth in the reference as well as the experimental period. The results of both procedures lead to aconclusion regarding the reduced biological excess sludge [t TS] during the period ofobservation.
Results at selected sewage treatment plants
Table 1 summarizes the experiences at the treatment plants in Germany currently operating withDOSFOLAT®XS.
Sewage treatment plants using DOSFOLAT®XS (as of May 6, 2004)
dry weather
Treatment plant
Sewage treatment

Chemical industry

(Lower Saxony)
Zweckverband Abwasser-
beseitigung Mittlerer Itzgrund
At three treatment facilities, the modification of the sludge discharge process is alreadyconcluded, so the first operational results can already be presented. The plants in question are - die industrial sewage treatment plant ISP at (Figure 4),- the middle east German MEG(Figure 5), and- The lowerSaxony Plant LSPFigure 6).
As previously explained, after the introductory phase for the stabilized folic acid, the dischargeof excess sludge is reduced stepwise each time by 10% at each sewage plant at predeterminedintervals. The magnitude of these intervals based on previous experience is between 3 and 7days and is presumably also dependent on the age of the sludge in the sludge activation systemin the period prior to dosing with the stabilized folic acid. It should be noted that the size of thedaily sludge discharge as "external intervention" in the system by operating personnel need notalways correspond to the daily production of excess sludge. With an equal load situation orconditions of the biocoenosis, a deviation of the daily sludge discharge from the actual daily incidence of excess sludge can thus lead to a decrease or increase in the solids content of theactivation tank, depending on whether more is removed than is produced each day or less. Adecrease or increase of the solids content can thius occur even with a change in the sludgedischarge process after the introductory phase (15 days) of stabilized folic acid addition if the10% reduction of the discharge at the prescribed interval does not reflect the actual incidence ofexcess sludge. The load data for the activation tank system (for example COD load,) as well asthe history of the solids content in the activation tank, are thus to be taken into account.
The effect of the stabilized folic acid on the daily production of excess sludge can be seen inFigures 4 through 6.
Figure 4: ISP sewage treatment plant - TOC load as activation load magnitude,
reduction of the excess sludge discharge as well as the equilibrium solids content in
the activation tank

At the ISP industrial sewage treatment plant, due to alkylphenolic residues and higer CODconcentration (average 1200 mg COD/l), the ongoing operating dosage of stabilized folic acidafter the initial phase was set at 0.2 ppm (usually this is done only with levels above 2,000 mgCOD/l, below that 0.1 ppm is typically used). During the conversion of the sewage dischargeregime of the ISP sewage treatment plant (Figure 4), a decline in the solids content wasinitially observed in the activation process. Later in the process, with increasing reduction of theexcess sewer discharge, the initial solids content is restored. The mean TOC load (TOC load) inthe observed period did not change and is within the usual fluctuation range for sewagetreatment plants. At the end of April the excess sewage discharge increased slightly, as thesolids content increased. At this point, more sewage was discharged than was produced on adaily basis. The time history of the operating parameters indicates that a “cautious approach” tothe newly adjusted equilibrium achieved by the stabilized folic acid between the excess sewagedischarge and the increase in excess sewage is required, in order to draw off only the excesssewage which is actually produced during a certain period.
The excess sewage discharge was reduced at the ISP sewage treatment plant by a total of 67%compared to the reference period without stabilized folic acid. Due to the fluctuation in solidscontent, in order to evaluate the reduction of the actual sewage produced compared to thereference period, a weight assessment which included the activation and the treated sewagedisposed of was necessary. This weight assessment indicated the reduction of excess sewage by30%.
In the plant trials with stabilized folic acid at the MEG and LSP sewage treatmentplants, a significant reduction of the excess sewage was also established (Figures 5 and 6).
These trials demonstrated that the solids content during the activation process in the modificationof the sewage discharge regime (reduction of the excess sewage discharge) and during thefollowing period remained constant for the most part, despite slightly increasing COD loads inthe feed to the biological process. The dimension of the daily sewage discharge as an “externalintervention” in the system is thus roughly identical with the daily production of excess sewagefor both systems. The solids content of the discharged excess sewage varied only slightly in theperiods under review, so that the reduced excess sewage discharge was approximately equal tothe targeted reduction in excess sewage (50% and 60% respectively) for both systems. Theaccompanying mass balances which were carried out also confirmed these results.
Figure 5: The MEG sewage treatment plant - COD load as activation load magnitude,
reduction of the excess sludge discharge as well as the equilibrium solids content in
the activation tank

Figure 6: The treatment plant LSP - COD load as activation load
magnitude, reduction of the excess sludge discharge as well as the equilibrium
solids content in the activation tank

In contrast to the ISP treatment plant, the age of the sludge in the MEG and LSP treatmentplants (both with aerobic sludge stabilization) prior to dosing with stabilized folic acid wasabove 30 to 40 days, whereas at Sasol it was significantly less than 20 days. At the ISP plantvariations of the solids content in the activation tank were documented during the investigations.
Similar results have been observed in the tests currently in progress at the sewage treatmentplant in Bavaria (the sludge age was also less than 20 days here before theaddition of stabilized folic acid). At the MEG and LSP facilities, however, nosignificant change in the solids content was observed. Whether this should lead to theconclusion that plants whose sludge is less than 20 days old will experience periodic changes inthe solids content (at otherwise equal loads) during the establishment of a new equilibrium statewhen compared with plants having older sludge, and whether these plants should have the timeinterval for 10% reductions of the excess sludge discharge extended to about 7 days instead ofthe 3 to 5 for plants with older sludge needs to be clarified through additional operationaltesting.
As part of operational testing with stabilized folic acid, changes in the biocoenosis weredocumented once a week by microscopic studies. It was found that the higher microorganismsdeveloped somewhat more slowly given the same feed of active biomass and the acceleration ofmetabolic processes in the sludge activation system, despite the fact that the age of the sludgewas doubled more or less as a result of reductions in the discharge of excess sludge.
Summary and future prospects
Due to its instability in aqueous solution, the vitamin folic acid is not present at high enoughlevels in the biocoenosis of treatment plants and therefore has a limiting effect on the metabolicprocesses of sludge activation. Folic acid is particularly important in regulating the 1-carbonmetabolism of the microorganisms and leads to an increase in metabolic activity.
DOSFOLAT®XS, which contains stabilized folic acid as its active ingredient, allows theexternal addition of folic acid to sewage treatment processes. The continuous dosing rate ofstabilized folic acid for moderate dry weather runoff triggered over a 24 hour period is designedto be 0.5 ppm initially ("shock dosing") and thereafter the operating dosage is 0.1 ppm.
According to the information currently available, evaluations of initial operation testing with thisproduct in Germany indicate that a reduction in excess sludge on the order of 45 ± 15% ispossible. No habituation has been observed in the process even after years of DOSFOLAT®use. DOSFOLAT®XS is available as a concentrate that is easy to administer and has aneffective delivered and has the effective power per kilogram of about 25 to 30 kg ofunstabilized folic acid.
Further investigations, for example for evaluation of the effects of stabilized folic acid on thereduction of the residual load in separated sludge water from sludge dehydration, increase of theprocess stability as well as release of additional tank capacity (reaction volumes) in facilitieswhose capacity is at full use or overloaded will be the object of future research projects.
Jörg Strunkheide, Dr. Ing.
IWB Institut Wasser und Boden e.V.
Oelgartenstrasse 18 † 53757 Sankt Augustin, Germany
Phone: +49 2241 341087 † Fax: +49 2241/334042

Kahle, K.: "Folsäure: Mangel im Überfluss?", term paper under the direction of Dr. P.
Schreier, professor for food chemistry, Bayerische Julius-Maximilians-Universität,Würzburg, 2003 Koebnick, C.: Giessen whole food nutrition study, Part 1: "Einfluss der Kostform aufden Vitamin B12- und Folatstatus in der Schwangerschaft", dissertation for the degreeof Doctor of Medicine, Giessen, 2000 Welch, A.D.; Null and void, C.A.: "Water-soluble vitamins concerned with one- andtwo-carbonintermediates", Annu. Rev. Biochem. 1952, 21, 633-686 Bässler, K.H.; Golloy, I.; Loew, D.; Pietrzik, K.: Vitamin-Lexikon für Ärzte,Apothekerund Ernährungswissenschaftler, 2nd Edition, Fischer Verlag, Stuttgart, 1997 Mitchell, H.K.; Snell, E. E.; Williams, R.J.: "The concentrations of 'folic acid'", J.
Chem. Soc. 1941, 63, 2284 Reuter, H.D.: "Grundlagen zur Pharmakologie von Folsäure", in: Folsäure-Mangel,Fachgespräch am 5. Juli 1986 in Rottach-Egern (ed K. Pietrzik), W. ZuckschwerdtVerlag, Munich - Bern - Vienna - San Francisco, 1987, 1-14 Anderl, A.: "Ein Vitamin für Kläranlagen", BIOTECHNOLOGIE 5,1987 N.N., 2004: experimental reports, masters and doctoral dissertations and publicationson the use of DOSFOLAT® from 1986 to 2004, Strunkheide, J.: "Überschussschlamm-Reduktion durch Tenside" in: wwt 12/2003, 34-41


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