Development of an organically certifiable growth medium for N-fixing cyanobacteria in a raceway biofertilizer production system

Abstract

The on-farm cultivation of N-fixing cyanobacteria in raceway ponds may provide an alternative N source in organic farming systems. The cultivation of cyanobacteria in an organic farming system requires an organically certifiable growth medium. Additionally, efficient cyanobacterial cultivation depends on production methods that reduce the severity of the three growth limiting factors present in outdoor raceway cultivation: inefficient solar irradiance, growth medium nutrient depletion, and day-night temperature fluctuations. The purpose of this work was two-fold, first to develop and test an organically certifiable growth medium, and secondly to test four specific production methods so as to optimize cyanobacterial growth and N-fixation. The four raceway production methods tested separately included: batch (B) versus semi-continuous (SC) operation mode, a culture depth of 20-cm versus 25-cm, bicarbonate supplementation in the growth medium, and four different cover plastics over raceways. All studies used a cyanobacterium cultured from a Fort Collins, CO lake, with 99% similarity to Anabaena cylindrica. Cyanobacterial growth was estimated by optical density (OD) and chlorophyll content and cyanobacterial N-fixation was estimated by net Total Kjeldahl Nitrogen (TKN). In chapter 2, "Comparison of cyanobacterial growth and nitrogen fixation in a newly developed organically certifiable growth medium and Allen and Arnon growth medium", a laboratory and raceway study were conducted. In the lab study, the nutrients of Allen and Arnon (AA) that were not organically certifiable were replaced with organically certifiable nutrients to compose the organic medium (RB). The exponential growth rate was significantly higher in the RB medium compared to AA. Conversely the net TKN in the RB medium was 37% lower than that of AA. The lower N-fixation in the RB medium was attributed to the presence of N in the P source used for RB medium (bone meal). In the raceway study, there was no significant difference in growth between the two treatments despite lower concentrations of P, Co, Zn, and B in the RB medium. An overarching limiting factor evident in both treatments such as light limitation or C depletion could explain why there was no observed growth effect due to the low P, Co, Zn, and B concentrations of RB medium. The net TKN between the two treatments was not statistically different, which suggests similar N-fixation. The conclusion of similar N-fixation was questioned due to the contribution of dissolved N from bone meal. Together, the studies support that the RB medium supports growth similar to that of the AA medium in raceway cultivation. However, since N was present in the RB medium, it is possible that maximal N- fixation was not achieved. Recommendations to increase nutrient concentrations in RB medium are discussed in chapter 4, "Future recommendations". In chapter 3, "Biomass yield and nitrogen fixation of cyanobacteria in outdoor raceways under batch versus semi-continuous operation", a SC treatment was operated under a 25% harvest regime every other day beginning on day 6. The B treatment was grown for 14 days, then 85% of the treatment was harvested and the remaining 15% was used as seed to begin a second B set. At the end of four weeks, biomass yield and total N fixed was calculated for the B and SC treatments. There was no difference in biomass yield or N yield between the two treatments. More than likely the SC was harvested when the culture density was above the optimal cell density range, resulting in a lower total biomass and N yield than what could have been achieved within the optimal cell density range. Determination of the optimal cell density and a specific harvest regime that maintains the SC within the optimal cell density would result in a higher total SC biomass and N yield compared to that of B. Possible experiments to determine the optimal cell density are discussed in chapter 4, "Future recommendations". In Appendix II, "Cyanobacterial growth and nitrogen fixation in response to depth, bicarbonate supply, and hoop house coverings in outdoor culture", three separate batch studies were conducted in 1.2-m (l) by 0.6-m (w) by 0.3-m (h) tanks. The first experiment compared the growth and N-fixation of batch cultures grown at two different depths (20-cm and 25-cm). Raceway depth did not have an effect on total growth or net N-fixation. The second experiment compared cyanobacterial growth and N-fixation in AA medium supplied with 0 mM (control), 0.2 mM (low treatment), and 2.0 mM (high treatment) of potassium bicarbonate (KHCO3). There was no increase in growth or N-fixation due to addition of KHCO3. It was concluded that inadequate KHCO3 was added to significantly increase growth and that the addition of NaHCO3 rather than KHCO3 is necessary to assure adequate Na concentrations needed for maximal bicarbonate uptake. The third experiment compared the growth and N-fixation of cultures grown under different hoop house plastics (Thermax, Luminance, Dura-film Super 4, and 4 mil Husky construction plastic) and a no-cover control. None of the covers tested in the study increased the growth compared to the no-cover control. Zn slowly leached from the cultivation tanks, so that by the end of the third study, Zn toxicity clouded the interpretation of results.