Microbial load in mass cultures of green algae Scenedesmus acutus and its processed powder

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1 J. Biosci., Vol. 3, Number 4, December 1981, pp Printed in Indi. Microbil lod in mss cultures of green lge Scenedesmus cutus nd its processed powder M. MAHADEVASWAMY nd L. V. VENKATARAMAN Centrl Food Technologicl Reserch Institute, Mysore MS received 3rd June Abstrct. Microbil contmintion in cultures of the lg, Scenedesmus cutus rised in outdoor open tnks nd lso in the processed powder of the lg ws monitored; The totl bcteril popultion incresed with time during the growth period of six dys. When combintion of molsses nd crbondioxide ws employed s crbon source for this lg, the bcteril lod incresed to 10 colony forming units/ml. Yest, molds nd lso coliforms were quntitted. Drum-drying the lge drsticlly reduced the bcteril lod nd storing the lgl powder for period of over 3 months did not increse the bcteril lod. Pthogens like Slmonell nd Stphylococcus were not detectble either in the open cultures or in the drumdried lgl powder. Although there re not set stndrds vilble in literture on the permissible level of the microbil contmintion in lgl biomss for use in foods, the microbil lod ppers to be within the limits of permissible levels stipulted by Indin Stndrd Institution stndrds for bby foods. Keywords. Green lg; Scenedesmus cutus; microbil lod; bcteril contminnts; processed powder. Introduction Alge re receiving wide ttention s source of biomss protein (BMP) for use in niml feeds nd foods (Mituy et l., 1953; Mteles nd Tennenbum, 1968; Clement et l., 1969; Dbh, 1970; Soeder nd Pbst, 1975; Becker nd Venktrmn 1978; Becker et l., 1976). The technology of lgl cultivtion nd processing is now known for few lge like Chlorell, Scenedesmus, Coelstrum nd Spirulin. Clen wter production of the green lg Scenedesmus cutus hs been stndrdized t this Institute (Venktrmn et l., 1977; Becker nd Venktrmn, 1978). In this procedure, the lg is mss cultured in open cultivtion bsins nd sterile conditions cnnot be mintined. Under these conditions, the culture my be contminted with heterotrophic bcteri even when it is grown utotrophiclly with simple inorgnic medi (Mituy et l., 1953; Leone. 1963; Blsco, 1965; Vel nd Guerr, 1966). The possible helth hzrds of such contmintion in mss lgl cultures for niml feed nd food cnnot be overlooked. While extensive investigtions hve been crried out on the nutritionl nd toxicologicl sfety of the lgl biomss Abbrevitions used: BMP, biomss protein; efu, colony forming units. 439

2 440 Mhdevswmy nd Venktrmn (Soeder nd Pbst, 1975; Becker et l., 1976; Subbulkshmi et l., 1976; Venktrmn et l., 1977b nd 1980), very little is known bout the microbil contminnts. No reports re vilble on the bcteril lod either in cultures or in processed mteril. The common occurrence of different species of bcteri in lgl cultures nd in processed products rises the question of their significnce in regrd to public helth. Microbiologicl guidelines or stndrds for processed lgl products re needed to ensure the qulity of the products nd to protect consumers from hzrds. The present study is n effort to determine the bcteril lod nd to quntitte nd identify the orgnisms in lgl cultures nd processed lgl powder. The study ws lso intended to focus ttention on the bcteriologicl spects of lgl biomss production. Mterils nd methods Cultivtion of lge Scenenedesmus cutus ws used in this study. Stock cultures were mintined on gr slnts. Inoculum for outdoor culturing ws prepred from slnts in flourescent light illuminted thermostts with light intensity of 5-7 K.lux, Air ws pssed into the culture tubes t rte of ml/min. From the culture tubes, the lge were trnsferred to glss crboys to build up enough inoculum for mss culturing outdoors. The detiled procedure hs been published elsewhere (Venktrmn et l., 1977; Becker nd Venktrmn, 1978). The lgl cultures both indoors nd outdoors were grown under non-septic conditions. The lgl cultures in outdoor tnks were concentrted by centrifuge seprtor nd dried on drier (electricl or stem heted) or sundried on plstic sheets. Growth Mesurements Algl growth nd consequently the biomss produced re expressed s mg of lge produced/litre culture. This ws computed by mesuring bsorbnce of the cultures t 560 nm nd converting the sme into lgl biomss with the help of Stndrd grph. The norml growth period of Scenedesmus cultures when it is redy for hrvest is bout 6 dys, nd hence ll mesurements were tken for this durtion lone. Storge The dried lgl biomss ws stored in polyethylene-lined luminium bgs nd kept for period of three months for storge studies. Smple preprtion For ll the ssys tht re reported here, 10 ml of lgl culture or 10 g processed lgl powder were used. The mteril ws mixed with 90 ml of 0.1% sterile peptone wter. This ws shken for 10 min nd suitble dilutions were mde for the vrious ssys.

3 Microbil lod in green lge 441 Determintion of vible count: Bcteri: Stndrd methods were followed to determine the plte counts using nutrient gr medi (Hrringen nd McCnce, 1976). Counts were expressed s colony forming units (cfu)/ml of culture or per g of lgl powder. Yest nd molds: These were ssyed on cidified potto-dextrose medium. The pltes inoculted with the smples were incubted t 25 C for 5 dys followed by counting. Coliforms: Coliform counts re expressed s most probble number using McConkey broth (Hrrigen nd McCnce, 1976). Differentil tests were used in the identifiction of the coliforms s Escherichi coli, Enterobcter erogens nd intermedite species (Speck, 1976). Stndrd methods were used for determining the cogulse positive Stphylococcus ureus (Thtcher nd Clerk, 1968). Suspected colonies were tested for cogulse ctivity. Anlysis for Slmonell ws crried out using the method of Thtcher nd Clerk (1968). Biochemicl tests The bcteril forms tht were quntitted on the totl plte counts were identified bsed on colony chrcteristics nd biochemicl tests s detiled in Bergey's mnul (Breed et l., 1957). Results Algl growth As cn be seen from tble 1, the growth of lg with the ddition of molsses lone ws lower thn with molsses nd CO 2. The level of molsses used in the study ppers to be optiml to prevent serious contmintion by bcteri. This is evidenced by the bsence of sugrs in the culture medium when tested the following dy fter the ddition of molsses. Bcteril lod The bcteril lod s function of the ge of cultures incresed both in indoor nd mss outdoor cultures. The increse ws much less in indoor cultures during inoculum development s compred to the open bsin lgl cultures exposed completely to the environment. The bcteril lod of cultures monitored t 0, 3 nd 6 dys of growth re summrized in tble 1. The contmintion is possibly from wter nd lso from ir. The excretion of certin orgnic substnces by the lgl cells into the medi which my support bcteril growth hs been reported in lgl cultures tht re grown in simple inorgnic medi (Vel nd Guerr, 1966). In indoor cultures, coliform group of orgnisms ws not detected. In outdoor cultures, bcteril lod incresed from 0.3 to /ml within 6 dys. However, there re some reports tht the bcteril levels remin stble without significnt increse in Chlorell cultures (Mituy et l., 1953; Leone, 1963). Detils of cultivtion methods nd the ge of cultures t which the bcteril mesurements were tken re not vilble for the bove nd hence this cnnot be explined.

4 442 Mhdevswmy nd Venktrmn Tble 1. Microbil lod in Scenedesmus cutus cultures rised indoors nd outdoors. Vlues in prenthesis indicte rnge Not detectble. The vlues represent men of five independent observtions. The coliform group of orgnisms ws found only in few btches. This type of contmintion cnnot be voided under the pttern of cultivtion used here. By suitble processing methods, they cn be eliminted in the finl product. Yest nd mold counts lso incresed in lgl cultures during the growth period of six dys. Effect of orgnic crbon source Sugrcne molsses is being considered s n lterntive or supplementry crbon source to CO 2, which is rther expensive. Addition of sugrcne molsses even t low level enhnces the bcteril lod in the cultures nd this my be ttributed to

5 Microbil lod in green lge 443 the presence of simple sugrs in the medium. It is lso possible tht bcteri my oxidize the smll mount of molsses rpidly nd liberte CO 2 into the medium, thus stimulting lgl growth. Mixotrophic cultures hve much higher bcteril lod ( cfu/6 dys growth) compred to heterotrophic cultures. This needs serious considertion s the lgl growth nd consequently the biomss productivity is higher in mixotrophic cultures. Pthogenic forms The pthogenic forms like Slmonell nd Stphylococcus were not found in the cultures t ny stge of growth of lgl cells. The mjor contminting orgnisms were identified by biochemicl tests of one hundred rndomly picked colonies. The results re listed in tble 2. Micrococcus species were more common thn those of Bcillus. Coliforms were much less frequent compred to other bcteril species. The vrious orgnisms of the coliform group identified re listed in tble 3. Tble 2. Bcteril forms isolted from outdoor open Scenedesmus cutus cultures Per cent occurrence of ech bcteril species out of 100 rndomly isolted colonies.

6 444 Mhdevswmy nd Venktmmn Tble 3. Identifiction of coliform from mss outdoor cultures of Scenedesmus cutus Per cent distribution of the vrious types mong one hundred rndomly selected coliform colonies. b Type I nd II clssified bsed on indole; methyl red; VP (Voges-Proskuer) nd Citrte tests. Effect of different methods of processing: Sundrying, nd drum-drying on electricl or stem heted driers hve been used to prepre lgl powder form the slurry. The microbil lod in the dried powder is much lower thn in cultures s would be expected (tble4). Drum-drying sterilizes the lgl cells t lest prtilly in spite of the short detention time (8-10 s). Drumdrying on electric dryer ppers to reduce the microbil lod nd yests nd molds more effectively thn stem-heted drier. This is probbly due to lesser drum temperture being ttined in the stem-heted drier. Tble 4. Microbil lod in dried lgl powder processed under different conditions Products were free from Stphylococcus, Coliform group nd Slmonell. Vlues represent men of five independent observtions. Figures in prenthesis indicte the rnge of vlues.

7 Microbil lod in green lge 445 When the cultures re grown mixotrophiclly with CO 2 nd molsses, the microbil lod is slightly higher thn those grown with CO 2 lone. This my be due to the presence of spore-forming bcteri nd lso due to higher microbil lod in the lgl culture itself (tble 1). The sun-dried lge hd much higher microbil lod thn drum-dried mteril. No Slmonell nd Stphylococcus were detected in the differently processed powders. Coliforms were totlly bsent in dried lgl powder even though they were present in lgl culture; possibly, they re destroyed during drying. The types of bcteril forms isolted from drum-dried lgl mteril re listed in tble 5. This differs significntly from the lgl cultures in the totl bsence of the vrious species of Micrococcus. Tble 5. Bcteril forms isolted from drum-dried Scenedesmus cutus powder. Per cent occurrence of ech bcteril species out of hundred rndomly isolted colonies. Storge studies Dt on storge studies conducted for 3-month period using drum-dried lgl powder re given in tble 6. There ws no increse in microbil lod. This my be due to low moisture content (6-8%) which is not suitble for bcteril growth. Tble 6. Microbil counts on stored dried lgl powder. Vlues represent men of three independent observtions. Temperture 28 ±2 C.

8 446 Mhdevswmy nd Venktrmn Discussion Microbil monitoring of lgl culture nd lgl powder is necessry, since it is likely to ffect the qulity nd sfety of the finl product. There re no interntionlly ccepted sfety limits tht re vilble to drw definite conclusion s to whether Scenedesmus cutus grown on pilot plnt scle t this Institute re within permissible limits. However, the Scenedesmus grown in clen wter nd the drum-dried powder hve microbil lods within probble sfety limits, when compred to Indin Stndrds Institute (ISI) guidelines for bby foods which is 50,000 colonies/g. The bsence of pthogenic forms like Slmonell nd Stphylococcus mkes the lgl powder sfe for utiliztion. Drum-drying of Scenedesmus is n bsolute necessity in order to rupture the undigestible cellulosic cell-wll for protein digestibility. This evidently gives n effective sterilizing effect, reducing the microbil lod. Sun-drying is not suitble method for processing Scenedesmus for the sme reson nd this hd been confirmed by erlier studies (Subbulkshmi et l., 1976). Scenedesmus cultures re lso contminted by other orgnisms like protozons, other lgl species nd wter insects nd this spect is not detiled here. However, it my be mentioned tht contmintion of Scenedesmus by these is very limited nd does not endnger either the culture or sfety in its use s food. The present world-wide emphsis hs been more on technologicl nd nutritionl spects connected with lgl biomss production. Though the possibility of using lge s supplementry food protein hs become less promising its use in feed is distinct possibility nd the bcteril lod is n importnt criterion for sfety of use. This should lso receive more ttention from the reserchers in the field. Acknowledgements This study ws crried out s prt of Indo-Germn collbortive work nd All Indi Coordinted Project on Alge of the Deprtment of Science nd Technology (DST), Indi. The uthors thnk Dr. R. Binsck, Germn Agency for Technicl Coopertion [GTZ], Eschborn, West Germny, Dr. W. E. Becker, Institut für Chemische Pflnzenphysiologie, Universitt Tübingen, Tubingen, West Germny, Mr. P. K. Rmnthn, Centrl Food Technologicl Reserch Institute, Mysore for their keen interest in this work. References Becker, W. E. nd V.enktrmn, L. V. (1978) Alge for feed nd food, A mnul on the cultivtion nd processing of lge s source of single cell protein, (Mysore: Wesley Press). Becker, W. E., Venktrmn, L. V. nd Khnum, P. M. (1976) Nutr. Rep. Int., 14, 305. Blsco, R. J. (1965) Appl. Microbiol, 13, 473. Breed, R. S., Murry, E. G. D. nd Smith, N. R. (eds) (1957) 7th edn. Bergey's mnul of determintive bcteriology (Bltimore: The Willims nd Wilkins Co) p. 455, 613. Clement, G., Durn-Chstel, H. nd Henry, V. (1969) Voeding, 30, 772. Dbh, R. (1970) Food Technol., 24, 659. Hrrigen, Ν. F. nd McCnce, Μ. Ε. (1976) Lbortory methods in food nd diry microbiology (New York nd London: Acdemic Press) pp. 132, 142, 258. ISI Stndrds for bby foods (1968) ISI: 1547, ISI Publictions, New Delhi. Leone, D. E. (1963) Appl. Microbiol., 11, 427.

9 Microbil lod in green lge 447 Speck, Μ. L. (1976) Compendium of methods for the microbiologicl exmintion of foods (New York: Americn Public Helth Assocition Inc.) Mteles, R. I. nd Tnnenbum, S. R. (1968) Single cell protein (Cmbridge: MIT Press). Mituy, Α., Nyunoy, Τ. nd Tmiy, Η. (1953) Algl cultures from lbortory to pilot plnt, (ed. J. S. Burlew) (Wshington: Crnegie Institute) p Soeder, C.J. nd Pbst,W. (1975) Production, properties, preclinicl nd clinicl testing of Scenedesmus ThePAGCompendium (New York: World Mrk Press Ltd.,) C-2, Subbulkshmi, G., Becker, W. E. nd Venktrmn, L. V. (1976) Nutr. Rep, Inst., 14, 581. Thtcher, F. S. nd Clerk, D. S. (1968) Microorgnisms in food (New York nd London: Acdemic Press). Vel, G. R. nd Guerr, C. N. (1966)J. Gen. Microbiol., 42, 123. Venktrmn, L. V., Becker, W. E. nd Shml, T. R. (1977) Life Sci., 20, 223. Venktrmn,L. V., Becker, W. E.,Khnum, P. M. nd Mthew, K. R. (1977b )Nutr. Rep. Int., 16, 231. Venktrmn, L. V., Becker, W. E., Rjsekrn, T. nd Mthew, Κ. R. (1980) Food Cosmet. Toxicol. 18,271.