THE BIOTECHNOLOGY OF EFFLUENT-GROWN Spirulina, AND APPLICATION IN AQUACULTURE NUTRITION THESIS Submitted in fulfilment of the requirements for the degree of MASTER OF SCIENCE (BIOTECHNOLOGY) Rhodes University Grahamstown by BRENTON ASHLEY MAART November 1992 TABLE OF CONTENTS ABSTRACT I LIST OF FIGURES IV LIST OF TABLES V1l1 CHAPTER 1 SALINITY AND ALGAL BIOTECHNOLOGY PART A MICROALGAE AND SALINE WASTEWATER 1 PARTB The Biotechnology of Spirulina PRODUCTION 10 CHAPTER 2 RESEARCH PROGRAMME Spirulina AND TANNERY WASTEWATER 27 CHAPTER 3 OCCURRENCE OF SpirulilUl IN TANNERY EFFLUENT 3.1 INTRODUCTION 30 3.2 RESEARCH OBJECTIVES 32 3.3 MATERIALS AND METHODS 33 3.4 RESULTS 34 3.5 DISCUSSION 43 CHAPTER 4 HARVESTING AND PROCESSING 4.1 INTRODUCTION 49 4.2 RESEARCH OBJECTNES 55 4.3 MATERIALS AND METHODS 55 4.4 RESULTS 61 4.5 DISCUSSION 62 CHAPTER 5 CHEMICAL COMPOSITION 5.1 INTRODUCTION 65 5.2 RESEARCH OBJECTIVES 68 5.3 MATERIALS AND METHODS 69 5.4 RESULTS 75 5.5 DISCUSSION 79 CHAPTER 6 TOXICOLOGICAL EVALUATION OF Spirulina BIOMASS 6.1 INTRODUCTION 84 6.2 RESEARCH OBJECTIVES 92 6.3 MATERIALS AND METHODS 92 6.4 RESULTS 98 6.5 DISCUSSION 112 CHAPTER 7 FEEDING Spirulina TO THE SOUTH AFRICAN ABALONE, HaIiotis midae 7.1 INTRODUCTION 120 7.2 RESEARCH OBJECTIVES 126 7.3 MATERIALS AND METHODS 126 7.4 RESULTS 131 7.5 DISCUSSION 136 CHAPTER 8 FEEDING Spirulina TO THE RAINBOW TROUT, Oncorhynchus mykiss 8.1 INTRODUCTION 141 8.2 RESEARCH OBJECTIVES 149 8.3 MATERIALS AND METHODS 150 8.4 RESULTS 157 8.5 DISCUSSION 165 CHAPTER 9 CONCLUSIONS AND RECOMMENDATIONS 172 REFERENCES 179 ACKNOWLEDGEMENTS 205 APPENDIX I 207 APPENDIX II 208 ABSTRACT The biotechnology of production and utilisation of the cyanobacterium Spirulina has been well documented. Research has centred mainly on application in human and animal nutrition, and has been motivated by the high protein, vitamin, fatty acid and growth factor contents. The main obstacle in realising the full potential of this feed source has been the high production costs associated with its mass culture in defined media. The observation of blooms of Spirulina in tannery effluent evaporation ponds in Wellington, South Africa, prompted this investigation into the harvesting, and nutritional and toxicological evaluation of this potentially low-cost production system, with the ultimate aim of using the product in aquaculture rations. An investigation of the chemical gradient along the evaporation cascade showed a positive correlation between the prevailing chemical conditions and the dominant species populations. A standing crop of 9.5 tonnes/ha of Spirulina was found to be present in the latter alkaline ponds, characterised by relatively lower organic and sulphur contents. Initial harvesting of the biomass was achieved by the design, construction and implementation of a small-scale screen harvest, which yielded a 25 kg (dry weight) crop. A scale-up model was then designed, and implemented in a technical scale harvest, yielding a crop of 250 kg (dry weight). Both these harvests utilised the bloom of surface-autoflocculated biomass. Concentrated cell slurries were sun-dried on muslin beds, and milled to a coarse powder. An evaluation of the harvest revealed a chemical content similar to other published reports of defined media cultures, with the exception of the protein and amino acid contents. The observed lower levels of the latter two are almost certainly due to the sun-drying method employed, known to reduce the protein content due to thermal denaturation. Legislation demands the strict toxicological evaluation of new protein sources, and because of the effluent-nature of the growth medium of this source of Spirulina, its viability lies only in the application as an animal feed or supplement. A range of toxicological tests were I chosen that were targeted to elucidate the possible toxicological constraints of this effluent­ grown source of protein in animal nutrition. The nucleic acid and pesticide contents of the harvested biomass were within the prescribed safety ranges. Atomic absorption showed minimal accumulation of minerals and heavy metals from the effluent. A bioassay with the brine shrimp Anemia salina showed that the biomass contained no toxicologically active water-soluble components. A short term feeding trial with new-born chicks showed that supplementation with Spirulina had no effect on the growth rates and feed conversion ratios of the different feeding groups. Pathological analyses showed that the liver was the only target organ to elicit a change in response to supplementation of the diets with Spirulina. A general decrease in liver weight was noted, with Cu, Ca, Fe and Zn being significantly accumulated. A histopathological examination however, showed no cellular and functional aberration from the control animals. The toxicological analyses gave the preliminary safe go-ahead for the evaluation of effluent­ grown Spirulina in aquaculture nutrition. The South African abalone Haliotis midae, and the rainbow trout Oncorhynchus mykiss were chosen as representative species of edible cultured organisms. The technology for the culture of the perlemoen abalone is being established in South Africa, with the main area of research being the development of an artificial diet for high density culture. A 40 day growth trial demonstrated that lower concentrations of Spirulina supplemented to an agar-based fishmeal diet resulted in growth rates and feed conversion ratios similar to the control fishmeal and purified-casein diets, and thus has application potential in the nutrition of this high-cost marine delicacy. The aquaculture technology of freshwater rainbow trout is already well established. An eight week feeding trial with various concentrations of Spirulina showed that this effluent-grown protein source can partially replace fishmeal in semi-purified diets. Fish fed Spirulina did not exhibit decisive manifestations of toxicity, as determined in a histopathological study. In addition, Spirulina supplementation resulted in enhanced colouration of the skin and flesh, which may have implications in the aesthetic marketing of this sought-after table fish. II The primary aim of this preliminary investigation thus concerned the determination of the biotechnological potential of this effluent-source of Spirulina. A technology transfer from the economically unfeasible defined-media culture was implemented. This project is ultimately aimed as a contribution towards the treatment of tannery wastewater, by the removal of contaminants from the effluent in the form of organic biomass. III LIST OF FIGURES Figure 3.1 Plan view of the evaporation ponding cascade at WTC, Wellington. Effluent is pumped from the factory to pond 1, flows to pond 5, where it is pumped to pond 6, and then concentrated by evaporation through to ponds 10 & 11. At certain times, ponds A and B remain stagnant, while the other nonds are used in the evaporation cascade. Figure 3.2 Red colour observed in the initial ponds in the effluent-treatment cascade. The colour is caused by the predominant halobacteria. Figure 3.3 Rafts of Spirulina in Pond D in the evaporation ponding cascade. Figure 3.4 Spirulina sp. in ponded effluent. Note the presence of the coexisting filamentous cyanobacterium, Anabaena spp. Figure 4.1 Diagrammatic representation of the small-scale screen harvester used for the concentration of Spirulina (holding reservoir, collecting reservoir, stilling baffle, screen, lateral supports, front supports, running groove). Figure 4.2 On-site operation of the screen harvester in a small-scale harvest of Spirulina biomass from tannery effluent. Figure 4.3 Side, plan and front elevation design of the technical-scale screen harvester for use at WTC, Wellington. Figure 4.4 Technical-scale harvest of the Spirulina biomass in pond D at WTC, Wellington. Figure 4.5 Sun-drying the Spirulina biomass on muslin beds. Figure 6.1 Survival of A. salina nauplii larvae at 24 hrs, after exposure to varying concentrations of Spirulina biomass. Figure 6.2 Survival of A.salina nauplii larvae at 12 and 24 hrs, after exposure to varying concentrations of lysed Spirulina biomass. Figure 6.3 Feed Consumption of the three feeding groups of chickens. Figure 6.4 Growth rates of the three feeding groups of chickens. Figure 6.5 Feed Conversion Ratios (FCR) for the three different feeding groups. Figure 6.6 Wet weight analysis of the hearts, livers, kidneys and spleens from the three chicken feeding groups. IV Figure 6.7 Dry weight analysis of the hearts, livers, kidneys and spleens from the three chicken feeding groups. Figure 6.8 Mineral and metal content of the dried liver samples as determined by atomic absorption spectroscopy. Figure 6.9 Sections of the hearts from the three chicken feeding groups (mag. - l00X). The myocardial fibres in all the examined samples spanning the three feeding groups appear to be faintly granular, but the change is mild and does not appear to be significant. No other significant lesions are noticeable in any of the specimens. (Myofibrils; Nucleus) Figure 6.10 Sections of the livers from the three chicken feeding groups (mag - 160X). There are no significant morphological lesions in the control and 10% feeding groups. One specimen from the 50% feeding group, however, exhibited atrophy of the hepatocyte chords, but this condition was not noted in any of the other specimens examined. Another specimen was shown to contain a few urate crystals in the lumen of some of the cortical tubes. This may be due to dehydration caused by the high concentration of NaCI in the biomass. Again, this condition was not noted in any of the other examined specimens. (Nuclei; Hepatocytes) Figure 6.11 Sections of the kidneys from the three chicken feeding groups (mag. = 160X). Generally, there are no significant lesions in any of the feeding groups. Some of the individual renal tubular epithelial cells in the 50% feeding group exhibit slight degeneration. This, however, is probably due to mild dehydration caused by the high NaCI concentration in the Spirulina biomass.