This 2-year project (Benemann et al. 1979; Eisenberg et al. 1980) extended from the end of 1977 to Winter 1979. Initially the approach to establish microstrainable cultures using the 12-m2 ponds, described in Section III. A.3., continued to be investigated. Essentially the same results as before were obtained: detention time was found to be the key environmental variable determining algal colony size (but not necessarily species composition) and a negative correlation was found between numbers of algal grazers and the large colonial algal types easy to harvest with microstrainers. Apparently the grazers preferentially consumed the smaller algae. Overall, the harvestability results with the microstrainers continued to be poor, so this line of research was abandoned during the initial period of this project.

Simultaneously with these studies, another project was being carried out under EPA sponsorship to study the settling of algae in the City of Woodland waste treatment ponds. This project used a “phase isolation” process, in which the algal cells were allowed to spontaneously settle when sewage inflow was stopped (Koopman et al. 1978, 1980). Although generally long times were required for this settling process (2-3 weeks), it was decided to investigate this general phenomenon of “bioflocculation” in high rate ponds. The process involved removing the algae from the paddle wheel-mixed ponds and placing them in a quiescent container, where they would spontaneously flocculate and rapidly settle. There are several apparently distinct mechanisms by which algae flocculate and then settle, including “autoflocculation”, which is induced by high pH in the presence of phosphate and divalent cations (Mg2+ and Ca2+), and flocculation induced by N limitation. Bioflocculation refers to the tendency of normally repulsive microalgae to aggregate in large flocs, that then exhibit a rather high sedimentation velocity. The mechanisms of bioflocculation involve extracellular polymers excreted by the algae, but the details remain to be investigated.

Settling tests were carried out with the cultures from the 12-m2 ponds. As with microstrainer harvesting, detention time and mixing velocity were the most important variables in promoting a bioflocculating culture. The rather rapid settling of many of the cultures was very encouraging. Also, the initial experiments with the 0.25-ha pond demonstrated a fairly rapid (<24 h) bioflocculation process.

At this time (mid 1978), the 0.25-ha pond had been divided into two 0.1-ha high rate ponds mixed with paddle wheels, and the bioflocculation-settling process using these pilot ponds became the focus of further research. Two 32,000 L settling ponds, with concave bottoms as deep as 2.5 m, were constructed to test bioflocculation-settling with the effluents of these ponds at the pilot scale. In initial experiments the two ponds were operated in parallel with two smaller ponds, at similar dilution rates (and with the only difference that one set of ponds was screened to remove larger grazers). The results (summarized in T able III. A.1.) were reasonably reproducible between the ponds. Algal solids removal through bioflocculation, as measured by 24-h settling in an “Imhoff Cone,” was high, about 90%.

Additional experiments, with the ponds operated in parallel at 2.5- and 5-day retention times (during September 1978) again resulted in removal efficiencies of over 90% for both ponds, including the 32,000 L settling tanks, based on measurements of suspended solids and chlorophyll. However, the shorter retention-time pond had more than twice the productivity of the longer-retention time pond (15 versus 7 g/m2/d). Bioflocculation was established as the method of choice for algal harvesting, as it seemed to be achievable even with high productivity cultures. Culture settleability was routinely determined during all the experiments with the high rate ponds. Table III. A.2. summarizes productivity and settleability for more than 1 year for the two 0.1-ha ponds. The 24-h laboratory settling tests correlated well with settling in the large (32­m3) settling ponds. Overall, a settling efficiency of greater than 85% was achieved, if the better of the two ponds was selected, without compromise in productivity. Annual gross productivity averaged almost 20 g/m2/d, estimated at about 90% algal solids. In many cases bioflocculation removals were higher than 95% on a chlorophyll a basis, indicating almost complete sedimentation of the algal biomass.

One factor that had a major effect on bioflocculation was mixing speed. At very low mixing velocities, the cultures settled very poorly. This is not unexpected, as any settling algae would have dropped out of the photic zone and been replaced by suspended cells in the absence of continuous mixing. However, a more fundamental investigation of the bioflocculation phenomenon (well reported in the ecological and some physiological literature) remains to be carried out.

Another activity carried out during this project was the cultivation of N-fixing microalgae using the supernatant of secondary growth ponds (see Figure III. A.3.). One problem was that culture collection strains of N-fixing cyanobacteria inoculated into the N-deficient ponds quickly succumbed. However, when the N-deficient wastewater samples were incubated in sunlight for a few weeks, N-fixing strains, which were indeed culturable, appeared spontaneously. One strain, an Anabaenopsis sp., was successfully cultured over long periods. However, the productivity of N-fixing cultures was only about half of what was observed with green algae under similar conditions. That is understandable because of the high energy requirement of N-fixation, which is not desirable in biomass fuel production. However, use of such algae for phosphate removal from wastewaters is of interest.

This project also investigated the anaerobic digestion of algal biomass using both a large (1,400- L) and several smaller (20-L) digesters. Algae provided a good substrate for anaerobic digestion, although not as good as conventional sewage sludge based on the conversion of organic C to methane. However, attempts to regrow algae on the effluent of the digesters resulted in poor productivities, as some unidentified factor appeared to limit growth on the regenerated nutrients. This was not investigated further, but clearly requires some attention in the future.

As part of this project, the energy requirements for high-rate pond operations, in particular mixing, were studied. The shaft power input required for mixing followed closely the predicted cube power law, demonstrating the need to keep mixing velocities below 30 cm/s to avoid excessive power inputs. At 15 cm/s, power inputs were only about 1 kWh/d (for the 0.1-ha

pond), increasing to about 10 kWh/d at 30 cm/s. Motor efficiency at the higher velocity (67%) was twice that at the lowest velocities, not a deciding factor. Clearly, mixing speeds higher than 30 cm/s are impractical, at least for energy production systems.

An energy analysis of such a microalgae wastewater treatment process was quite promising, with the wastewater system being at least energy self-sufficient based on the requirements for sewage and algal pond effluent lift requirements, as well as algal pond mixing and other requirements. Such systems should be able to generate a net positive energy output if operated to maximize algal biomass through the addition of CO2 (Figure III. A.3.).

Overall this project marked a major advance in this technology, as it demonstrated at the pilot plant scale a relatively low-cost and reasonably reliable microalgae harvesting process that does not unduly interfere with other objectives, in particular microalgae productivity. (See Benemann et al. [1980] and Eisenberg et al. [1980].)