Rey. Biol. Trop.,43(6):595c600, 1998

Population dynamics ofBrachionus calyciflorus (Rotifera: } in waste water frmIl food-processing industry in Mexico

Raymundo Alfredo Arévalo Stevenson, S.S.S. Sarma* andS. Nandinil Carrera de Biología, UNAl\lCCarnpus.Iztacala, AP 314 CP 54000, Los Reyes, lztacala, Tlanepantla, Edo. de México, México. Telefax: +52 (5) 623 1212, E-Mail: [email protected] *Corresponding author.

Receiyed 26-IX-1997. Correcled 15-IV-1998. Accepted 29-IVc1998.

ADst¡:a,ct:Waste water from Mexico's largesl food processingindustrial sector (basedonmaize, nejayote water) was �sed ior growingBrachionus calyciflorus isolated from Lake Chapultepec inthe Federal District of Mexico.(D.F.). Nejayote waterwa s c(jllected from Colonia Providencia, D.F. Experiments wereconducted al 25°Cin 25 mI capacity "ialswith20 mIof medlUminto which we intr�ducedB. calyciflorus ataninitial density of � ind mI-l. Theexperi­ W\1ental designconsisteg a total 003 testves.sels (2 food óombinations X 5densities X 3 replicates = 30 plus 3 repli­ cates as colitrols tha! contaiped only¡¡lgae). Experi rp.entsW"re tenninated <ÚterdaY16,Wastewatetin original concen- i tr¡uÍoll did no! supportrQtifets.Jloweyer, whenqi1uted t? 5 con¡:entrations (ranging Crom 2% to 32% andpH adjusted . t07.0), density increase� \Y¡thincreasingconc¡:nt ration oí wastewater, Green a1gae (at COf¡stant density of 2 X 6 1 .o ceIIsmr � .' of Chlordla) in �Ombination w¡ih was�.waterresl}ltedi �a higher abundance 0f only al higher 1 (;oncentrations (above 8%}ofwaste water:The mrudmutn peakdensityof rotifers (238 ± 5.0 ind ml- ) was obtained at 16% dilution of waste water (\nd with addition.of Chlorella. Therate ofpopulation ¡ncrease per day (r) (mean±SD) varied from .0.355 ± .0. .059 to .0.457. ±0..o48 deper¡dingonfoodcom binatioll and concentration.

. R. otifer.,B.rachionus,waste water, Key .words: .popul:¡1i.(Í�, '"growt!)¡ ,�,, M' ,'

Maize, particularlyin the form oí tortilla (a p¡:¡rtlyhydrúlizesthe hard seedcpat andturnsthe kind of thin-plate like soft dry bread) ís one of seedsinto softeasilyq:u.shableil1\lterial to fmm the most important components in the dictof dough . for tort.illa man\lfacture. Dunng tbis the Mexican population, Tortitla industry process of boiling, anumber of organic (both ranges from large scale (up to 50 tons per day) particulate and.dissolved) sllbstances including to cottage levelproductlon (about 700 kgs per proteins, carbohydrates andlipids are lost into day)(Durán de Bazúa 1988): �orethan 95% of thewater, resulting in an organically rich medi­ the tortilla industry in. Mexico utilizes maize um (Pedroza1985). (and the re�t 4tilizes wbeat). For preparing tor­ Rotifers a.re known. to utílize particulate tilla bythe.ttaditipnal method caHed nixtamal­ organic matter directly (pQurriot 1965) anddis­ iZ\lüon (lime-steeping. ofcorn), the.maizeseeds solved organicmatter via bacteria and protozoa are pre-boiled in wate,rwitblime. This process (Arndt 1993). They thus form an important link DE 596 REVISTA BIOLOGIA TROPICAL

in the aquatic food chain, particuIarly in trans­ selected a site (Street: Estado de Zacatecas and fering energy from lower to higher trophic lev­ Estado de MoreIos, Colonia Providencia) in the els. A number of workers have obtained high Federal Distríct of Mexico which regularly production of rotifers (up to 500 ind mrl) in receives the nejayote water. Every alternate day outdoor aquacultural practices using piggery waste water was collected from the sarue site wastes and liquid organic wastes (Jhingran and stored in a refrígerator for the experiments. 1991). We did not use waste water which was stored for Rotifers of the genus have been more than 2 days. Since the initial pH of the widely used as starter food for rearing larval nejayote water was as high as 9.0 due to high fish and crustaceans in aquaculture (Lubzens et levels of lime (which does not support rotifer al. 1989), indicators of pollution (SIadecek growth (Mitchell and Joubert 1986», it was 1983) and as bioassay organisms (Snen and brought down to 7.0 by the addition of diluted Janssen 1995). They have also been included as HCl. This process resulted in sorne precipitation standard bioassay organisms by the American at the bottom of the vessel, which was siphoned Socíety of Testing and Materials in the USA off. Since Brachionus does not feed effectively (Anon. 1991). Since the waste water of tortilla on particles larger than 20 �m in size (Poumot industry (known as nejayote water) is rich in 1965), we filtered this water using 20!-lm mesh organic matter, we aimed at testing its suitabili­ and the supernatant was diluted as required. This ty for growing rotifers. process also helped in removing most ciliates in the waste water. Once adjusted, the pH of the medium did not vary over a 24 h periodo Based MA'IERIALS AND METHODS on prelíminary tests it was observed that B. caly­ ciflorus did not grow well directly on the waste The rotifer Brachionuscalyciflorus (average water at its original concentration. We therefore adult length excluding spines = 185 ± 12 !-1m) diluted this water (using EPA medium) to vari­ was originally isolated from Lake Chapultepec ous concentrations up to 32% (= 68% dilution) (Mexico City) and successfully cultured in the which permitted the rotifers to grow for a rea­ laboratory using the single-celled green algae sonable periodo To estimate the growth of B. (Chlorella vulgaris, average cell diarueter: 5.48 calyciflorus, we chose 5 concentrations of the ± 1.21 !-1m) as the exclusive food (Sarma et al. waste water viz. 2, 4, 8, 16 to 32%. Growth of 1997). Chlorella was mass cultured using Bold the rotifer on the waste water was tested, both Basal medium (Borowitzka and Borowitzka with and without an additional food source 1988). (Chlorella at 2 X 106 cells m¡-l). Our stock as well as mass cultures of rotifers For experiments, we used 25 mI capacíty were maintained in EPA medium (Anon. 1985) transparent vials containing 20 mI of the waste androutinely fed daily the green algae at a densi­ water at the desired concentration and combina­ ty of approximately 2 X 106 cells mrl . Although tion of waste water. AH experiments were con­ in our routine cultures we were able to obtain ducted in thermostatically controlled water­ rotifers at a density of about 100 ind rnl-I; we baths set at 25 oc. The initial pH of the medium generally maintained the population below 50 ind was adjusted to 7.0. For each dilution of waste mr 1 in order to reduce thepossibility of male pro­ water, we used 3 replicates. In combinations duction. For regular feeding as well as for exper­ where alga was used as an addítional food 6 ¡ments, we used log phase algae, centrifuged at source, íts concentratíon was kept at 2 X 10 4000 rpm, rinsed in distiUed water and resus­ cells m¡-l. Thus, the experimental design con­ pended in EPA medium. The density of algae was sisted of 33 test vesseIs (2 food combinations X estimated using a haemocytometer. 5 densities X 3 replicates = 30 plus 3 replicates For obtaining a constant source of waste as control which contained only algae at the water from tortilla processing industry, we same density). lnto each of the test vessels, we AREVALO eral.: Population dynamicsófBráchionus calyciflorus 597

introduced B. calyciflorus at a density of 1 ind mI-l. The initial popuJation ofrotifers, counted 80 Control (0%) individuaUy, consisted of actively reproducing females of mixed age-group obtained from a 60 mass-culture tank during the exponential phase of their growth. The test vessels were main­ tained in diffuse and continuous fluorescent 40 iIlumination. Aeration was provided to the test vessds in arder to keep the food-párfiCles úHhe

test medium in suspension. For counting rotifers, we used one of the two

methods: a) whole count when the density of o rotifers was less than 5 ind mr1 or, b) aliquot O 2 4 6 8 10 12 14 16 subsamples of 1-5 mI volume whenthedensity 50 was greater than 5 ind mI-l. Fore�ch replicate, � 2% .- we counted at least 3 subsample5j.Following the . inoculatíon, we estimatedthe popuJation densi­ E 40 -t:i ty every day until most replicates completed §. one population Thus, the experiment was 30 cyc1e. �U) c: tenni�iltedafter day16. Everyday, after esti­ Q) "O matihg".the'populatioll density, rotifersftom' all c: 20 o repli2&,eswere transferred to fresh rnediUm :; "5 with appropriately diluted ""aster water and a. o 10 foodcombination. o.. For,estimating the pppulation density, vie counted onIy Iive female rotifers.Population dens1ty of rotifers was expressed,as num:ber per mLFor estimating the populationgrowth I-a,te (r)of totifers, we used the following fórmula:

1'= Nc nNo)/t (In l . where No :¡nitial population den:sity Nt = ¡:>opulatlon densit)' afrerthe,time t t= tirtte in �ays

RESULTS

2 4 6 810 12 14 16 In general the density of Brachionus caly­ Time (days) ciflorus increased with incteasing concentra­ HonoLwaste water in themedil.ltn. The popu­ latíon density of the rotifers grownin waster

water with additíon of algae showed a similar Fig.l. Population growth. of ¡he .totifer B. calyciflorus in trend(Figs 1 & 2). Anincrease in the proP9r­ relationto differentGoncentratlons ofwaste waterand in the presence andahsence ofthealgae ehlarel/a(at 2 X I06cells tion . of w�ste water in the mediu1l1 beyond mI-l). Shown ate the mean±SE values based on tbree replí· 16% did not suppqrt high population density cate recordings. Concentration of nejayote water is rotifers. of expressed in percentage. 598 REVISTA DE BIOLOGIA TROPICAL

140 300 8% - Presence � A 120 ";" = Absence _____ Presence E 250 ___ 100 Absence "'Óc: ;;;.. 80 � 200 c: (\) 60 "t:I .§ 150 40 (¡j "S c. 20 8. 100 E O ::J E O 2 4 6 8 10 12 14 16 'x <11 50 :E

� 300 ....., O E 250 "'Ó O 2 4 8 16 32 é Concentration of the waste water (%) � 200 '"c: (\) "t:I 150 r c: 0.55 o - Presence B :¡; :s = Absence "S 100 >- c. <11 o "t:I 0.50 ,.... o.. 50 a;c. CI) U) t- <11 0.45 2 4 6 � .5 c: 0.40 r-TI 60 o 32% ii I "S c. o 50 c. 0.35 'O

40 2<11 o:: 0.30 30 0.25 20 O 2 4 8 16 32 Concentration of the waste water (%) 10

2 4 6 8 10 12 Fig.3a. Maximum population density reached by Time (days) Brachionus calyciflorus in the presence and absence of the aIgae Chlorella(at 2 X 106 cells mI-1) at different concen­ trations of the waste water. Shown are the mean ± SE vaI­ ues based on three replicate recordings. Fig.2. Population growth oC the rotiCer B. calyciflorus in Fig. 3b. Rate of population growth per day (r) oC relation to different concentrations oC waste water and in the Brachionus calyciflorus in the presence and absence oC the presence and absence oC the aIgae Chlorella (at 2 X 1 ()6 aIgae Chlorella (at 2 X 106 cells mI-1) at different concen­ cells mI-1). Other details as in Fig. . 1 trations oC the waste water. Shown are the mean ± SE vaI­ ues based on three replicate recordings. AREVALO et al.: Population dynamics of Brachionus calyciflorus 599

The influence of algal food was strongly evi­ 3 ind ml-l, respectively) were less. The addition dent in media with higher percentage of waste of algae was useful at 8% and 16% concentra­ water. The maximum peak density of rotifers tion. A further ¡ncrease in the concentration of (241 ± 48 ind mrl) was obtained at 16% dilu­ the nejayote water resulted in a prolonged lag tion of waste water and with addition of phase after which, however, rotifers grew but Chlorella (Fig. 3a). TIlÍs variable was signifi­ not exactly comparable to the control with algae cantly affected by the food combination and the alone. The addition of algae at higher concentra­ concentration of waste water and their interac­ tions of waste water may have helped in absorb­ tion (P < 0.001, 2-way ANOVA). ing or even utilizing the toxÍG dissolved sub­ The rate of population increase per day (l') stances (such as dissolved ammonia and other varied from 0.355 ± 0 .059 to 0.457 ± 0.048 nitrogen-containing inorganic compounds) depending on food combination and concentra­ (Borowitzka and Borowitzka 1988). tion (Fig. 3b). The r value was significantly The population density of Brachionuscalyci­ affected by both, the addition of algae in the floras obtained here indicated that nejayote medium and the concentration of waste water (P water at 8% concentration had utilisable organic < 0.001, 2-way ANOVA). The interaction of load comparable to 2 X 106 cells m¡-l of food concentration and combination also had a Chlorella biomass. In terms of dry weight, it is significant ímpact on the population growth rate equivalent to about 28.4 fJg mI-l. This implied (P < 0.001, 2-wayANOVA). The influence of that the undiluted waste water may have readily algal food on the rate of population increase usable organic matter higher than 355 mg r1 was not significant at 2% and 4% concentra­ which could have a potential to result in the pro­ tions of the waste water (P > 0.05, F-test). duction of about 1000 ¡nd ml-1 of rotifers. Production of rotifers of this density is not uncommon under field conditions. For example, DISCUSSION piggery wastes and farmyard manures added to the fish tanks yield rotifers at a density compa­ Our study confirmed the presence of utiliz­ rable to. this (Jhingran1991). able form of organic matter in the nejayote The rate of population increase in rotifers water since rotifers grew well in the absence of observed here in two foodcombinations and 5 den­ algae. We analyzed the waste water directly for sities is within the range known for this· . the presence of zooplankton particularly For example, S anna et al. (1997) have reported r rotifers. We did not notice any zooplankton values for this species in the range of 0.2- 0.8 (except sorne ciliates) suggesting that the nejay" depending on !he foad concentration and inocula­ ote water as such is not suitable for growing tion density. In rare cases, ther value ofthisspecies rotifers. This was evident from the Fig. 2 in exceeds 2.0 (Bennett and Borass 1989). which when the waste water concentratíon was Our findings suggest the possible utilization 16% or 32%, rotifers did not maintain a popula­ of waste water from tortilla industry for the pro­ tion beyond a few days. One of the main rea­ duction of rotifers, which can be utilized in sons why nejayote water cannot be directly used aquaculture as starter food for rearing fish and is the high concentratíon of particulate organic crab larvae. Since the nutritional quality of matter.At high concentration of particulate mat­ waste water from the tortilla industry does not ter, generally, !he feeding and conversion effi­ vary significantly (Pedroza 1985), these results ciencies decrease (Downing and Rigler 1984). may be applicable for nejayote water col1ected Thus, when the waste water concentration was from other sÍtes. The present study can also be a 8%, the density of rotifers grown in the absence start-point for other studies such as those on of Chlorella was 77 ± 3 ind mJ-l, but when the nutritional aspects of rotifers produced using the concentrations were 16% and 32%, the nejayote water. observed maximum densities (61 ± 12 and 29 ± 600 REVISTA DE BIOLOGIA TROPICAL

ACKNOWLEDGMENTS Bennett, W. N. & M. E. Borass. 1989. A demographic pro­ file of the fastest growing metazoan: a strain of Brachionuscalycijlorus (Rotifera). Oikos 55: 365-369. One of us (SSSS) is thankful to CONACyT

(SNI) grant (No. 18723). RAAS thanks C. Borowitzka, M. A. & L. J. Borowitzka. 1988. Micro-algal Carlos Delgado for granting study leave. biotechnology. Cambridge University, London. 477 p.

Downing, J. A. & F. H. Rigler (eds.). 1984. A manual on methods for the assessment of secondary productivity RESUMEN in freshwaters. Blackwell Scientific, Oxford. 501 p.

Se usó aguas de desecho de la industria de la masa y la Durán de Bazúa, C. 1988. Una nueva tecnología para la tortilla (aguas de nixtamal o nejayote) para crecer rotíferos extrusión alcalina de maíz y sorgo. Monografia Brachionus calycijlorus. de agua dulce, El nejayote sin dilu­ Tecnólogica 2. Universidad Nacional Autónoma de ción no permitió el desarrollo del rotífero. Sin embargo, México. Mexico D. F., 71 p. cuando se diluyó, B. calycijlorus aprovechó la materia

orgánica. El agua de desecho por sí misma (sin alimento Jhingran, v.G. 1991. Fish and fisheries of India. Hindustan algal adicional) fue comparable al agua con densidades del Pub\., New Delhi, 727 p. I alga Chlorella de 2 X 106 células m¡- . Concentraciones de nejayote por encima del 8% no permitieron el crecimiento Lubzens, E., A. Tandler & G. Minkoff. 1989. Rotifers as poblacional. Sin embargo, la presencia de alga permitió el food in aquaculture. Hydrobiologia 186/187: 387-400. desarrollo del rotífero a concentraciones de 8% y 16%.

Nuestros hallazgos indican que el nejayote puedo ser uti­ Mitchell, S.A.& H. B. Joubert. 1986. The effect of elevated lizado de manera efectiva para la producción de rotíferos a pH on the survival and reproduction of Brachíonus acuacultura. gran escala para la calyciflorus. Aquaculture 55: 215-220.

Pedroza, R. 1985. Estudio de la degradación biológica de los efluentes de la nixtamalización. Tesis de maestría, REFERENCES Universidad Iberoamericana México D.F., Mexico. 75 p.

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