Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris

We tested 10 different Chlorella and Parachlorella strains under lipid induction growth conditions in autotrophic laboratory cultures. Between tested strains, substantial differences in both biomass and lipid productivity as well as in the final content of lipids were found. The most productive strain (Chlorella vulgaris CCALA 256) was subsequently studied in detail. The availability of nitrates and/or phosphates strongly influenced growth and accumulation of lipids in cells by affecting cell division. Nutrient limitation substantially enhanced lipid productivity up to a maximal value of 1.5 g l⁻¹ day⁻¹. We also demonstrated the production of lipids through large-scale cultivation of C. vulgaris in a thin layer photobioreactor, even under suboptimal conditions. After 8 days of cultivation, maximal lipid productivity was 0.33 g l⁻¹ day⁻¹, biomass density was 5.7 g l⁻¹ dry weight and total lipid content was more than 30% dry weight. C. vulgaris lipids comprise fatty acids with a relatively high degree of saturation compared with canola oil offering a possible alternative to the use of higher plant oils.     

Energy Demands of Nitrogen Supply in Mass Cultivation of Two Commercially Important Microalgal Species, <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Dunaliella tertiolecta</Emphasis>

Mass culture of microalgae is a potential alternative to cultivation of terrestrial crops for bioenergy production. However, microalgae require nitrogen fertiliser in quantities much higher than plants, and this has important consequences for the energy balance of these systems. The effect of nitrogen fertiliser supplied to microalgal bubble-column photobioreactor cultures was investigated using different nitrogen sources (nitrate, urea, ammonium) and culture conditions (air, 12% CO₂). In 20 L cultivations, maximum biomass productivity for Chlorella vulgaris cultivated using nitrate and urea was 0.046 and 0.053 g L⁻¹ day⁻¹, respectively. Maximum biomass productivity for Dunaliella tertiolecta cultivated using nitrate, urea and ammonium was 0.033, 0.038 and 0.038 g L⁻¹ day⁻¹, respectively. In intensive bubble-column photobioreactors using 12% CO₂, maximum productivity reached 0.60 and 0.83 g L⁻¹ day⁻¹ for C. vulgaris and D. tertiolecta, respectively. Recycling of nitrogen within the photobioreactor system via algal exudation of nitrogenous compounds and bacterial activity was identified as a potentially important process. The energetic penalty incurred by supply of artificial nitrogen fertilisers, phosphorus, power and CO₂ to microalgal photobioreactors was investigated, although analysis of all energy burdens from biomass production to usable energy carriers was not conducted. After subtraction of the power, nitrogen and phosphorus energy burdens, maximum net energy ratios for C. vulgaris and D. tertiolecta cultivated in bubble columns were 1.82 and 2.10. Assuming CO₂ was also required from a manufactured source, the net energy ratio decreased to 0.09 and 0.11 for C. vulgaris and D. tertiolecta, so that biomass production in this scenario was unsustainable. Although supply of nitrogen is unlikely to be the most energetically costly factor in sparged photobioreactor designs, it is still a very significant penalty. There is a need to optimise both cultivation strategies and recycling of nitrogen in order to improve performance. Data are supported by measurements including biochemical properties (lipid, protein, heating value) and bacterial number by epifluorescence microscopy.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Outdoor open thin-layer microalgal photobioreactor: potential productivity

We have previously estimated the productivity and photosynthetic efficiency of the microalga Chlorella sp. grown in an outdoor open thin-layer photobioreactor under climate conditions typical of the Middle European region, i.e. with many days unsuitable for intensive growth of algae (cloudy and rainy days, low air temperature, low solar PAR input).To estimate the real potential productivity of the bioreactor, we collected data on algae yields obtained during clear summer day periods. Cultivation was performed in fed-batch cycles in a bioreactor with a 224 m² culture area (length 28 m, slope 1.7%), and a 6–7 mm-thick layer of algal culture. The suspension volume in the bioreactor was 2,000 L. The mean values found for Třeboň (49°N), Czech Republic, as an average of several sunny summer cultivation periods in July, were: net areal productivity, P _net = 38.2 g dry weight (DW) m^-2 day^-1; net volumetric productivity, P_vol, = 4.3 g algal DW L^-1 day^-1, photosynthetic efficiency (based on PAR), η_net = 7.05%. The peak values were: P _net about 50 g (DW) m^-2 day^-1, η_net about 9%. Algal growth rate was practically linear up to high biomass densities (40–50 g DW L^-1, corresponding to an areal density of 240–300 g DW m^-2), at which point the culture was harvested. The concentration of dissolved oxygen increased from about 10 mg L^-1 at the beginning to about 23 mg L^-1 at the end of culture area at noon. Use of the above-described technology for economical production of bioethanol is proposed.     

Continuous production of isopropanol and butanol using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> DSM 6423

Clostridium beijerinckii DSM 6423 was studied using different continuous production methods to give maximum and stable production of isopropanol and n-butanol. In a single-stage continuous culture, when wood pulp was added as a cell holding material, we could increase the solvent productivity from 0.47 to 5.52 g L⁻¹ h⁻¹ with the yield of 54% from glucose. The overall solvent concentration of 7.51 g L⁻¹ (39.4% isopropanol and 60.6% n-butanol) with the maximum solvent productivity of 0.84 g L⁻¹ h⁻¹ was obtained with two-stage continuous culture. We were able to run the process for more than 48 overall retention times without losing the ability to produce solvents.     

Heterotrophic production of Chlorella sp. TISTR 8990—biomass growth and composition under various production conditions

The green microalga Chlorella sp. TISTR 8990 was grown heterotrophically in the dark using various concentrations of a basal glucose medium with a carbon‐to‐nitrogen mass ratio of 29:1. The final biomass concentration and the rate of growth were highest in the fivefold concentrated basal glucose medium (25 g L^?1 glucose, 2.5 g L^?1 KNO₃) in batch operations. Improving oxygen transfer in the culture by increasing the agitation rate and decreasing the culture volume in 500‐mL shake flasks improved growth and glucose utilization. A maximum biomass concentration of nearly 12 g L^?1 was obtained within 4 days at 300 rpm, 30°C, with a glucose utilization of nearly 76% in batch culture. The total fatty acid (TFA) content of the biomass and the TFA productivity were 102 mg g^?1 and 305 mg L^?1 day^?1, respectively. A repeated fed‐batch culture with four cycles of feeding with the fivefold concentrated medium in a 3‐L bioreactor was evaluated for biomass production. The total culture period was 11 days. A maximum biomass concentration of nearly 26 g L^?1 was obtained with a TFA productivity of 223 mg L^?1 day^?1. The final biomass contained (w/w) 13.5% lipids, 20.8% protein and 17.2% starch. Of the fatty acids produced, 52% (w/w) were saturated, 41% were monounsaturated and 7% were polyunsaturated (PUFA). A low content of PUFA in TFA feedstock is required for producing high quality biodiesel. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1589–1600, 2017     

Factors affecting lipid accumulation by <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> in a two-stage cultivation process

Reserve lipids of microalgae are promising for biodiesel production. However, optimization of cultivation conditions for both biomass yield and lipid production of microalgae is a contradictory problem because required conditions for both targets are different. In this study, a two-stage cultivation strategy is proposed to enhance lipid production of the microalga Nannochloropsis oculata. Biomass growth and lipid production were carried out in two separate and non-interacting stages. In first-stage cultivation, microalgae were cultivated in optimal conditions for cell growth. Then, microalgae were harvested and transferred into a growth-limited environment, thus enhancing lipid production of microalgae. Here, optimization of the lipid production stage (second stage) with respect to different levels of inoculum concentration, salinity of culture broth, and intensity of irradiance was performed. The results show that irradiance exhibits a significant influence on lipid production. The highest lipid productivity of 0.324 g L⁻¹ day⁻¹ was obtained with an inoculum concentration of 2.3 g L⁻¹, a salinity of 35 g L⁻¹, and an irradiance of 500 μmol photons m⁻² s⁻¹. The final yield of lipid obtained from the two-stage process was 2.82-times higher than that from traditional single-stage batch cultivation systems.     

A novel photobioreactor with transparent rectangular chambers for cultivation of microalgae

An open tank photobioreactor containing transparent rectangular chambers (TRCs) was developed to improve the photosynthetic efficiency of microalgal cultivation. The TRCs, made of transparent acrylic, conducted light deep into the photobioreactor, especially at high cell concentrations. The average irradiance, I_av, was calculated by Lambert–Beer's law, and was used to determine the light conditions in the cultivation system. The photobioreactor provided large areas of illumination that improved the effective utilization of light energy for microalgae growth and created a good artificial environment for a high rate of cell growth, even at low I_av. The biomass concentration of Chlorella sp. reached 3.745 g L⁻¹ on the 13th day, with biomass productivity of 0.340 g L⁻¹ d⁻¹. The total biomass obtained was 56% more than that of similar culture systems without TRCs.     

Productivity correlated to photobiochemical performance of <Emphasis Type="Italic">Chlorella</Emphasis> mass cultures grown outdoors in thin-layer cascades

This work aims to: (1) correlate photochemical activity and productivity, (2) characterize the flow pattern of culture layers and (3) determine a range of biomass densities for high productivity of the freshwater microalga Chlorella spp., grown outdoors in thin-layer cascade units. Biomass density, irradiance inside culture, pigment content and productivity were measured in the microalgae cultures. Chlorophyll-fluorescence quenching was monitored in situ (using saturation-pulse method) to estimate photochemical activities. Photobiochemical activities and growth parameters were studied in cultures of biomass density between 1 and 47 g L⁻¹. Fluorescence measurements showed that diluted cultures (1–2 g DW L⁻¹) experienced significant photostress due to inhibition of electron transport in the PSII complex. The highest photochemical activities were achieved in cultures of 6.5–12.5 g DW L⁻¹, which gave a maximum daylight productivity of up to 55 g dry biomass m⁻² day⁻¹. A midday depression of maximum PSII photochemical yield (F _v/F _m) of 20–30% compared with morning values in these cultures proved to be compatible with well-performing cultures. Lower or higher depression of F _v/F _m indicated low-light acclimated or photoinhibited cultures, respectively. A hydrodynamic model of the culture demonstrated highly turbulent flow allowing rapid light/dark cycles (with frequency of 0.5 s⁻¹) which possibly match the turnover of the photosynthetic apparatus. These results are important from a biotechnological point of view for optimisation of growth of outdoor microalgae mass cultures under various climatic conditions.     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Production of Cold-Adapted Amylase by Marine Bacterium <Emphasis Type="Italic">Wangia</Emphasis> sp. C52: Optimization,Modeling, and Partial Characterization

The aim of this work was to optimize the fermentation parameters in the shake-flask culture of marine bacterium Wangia sp. C52 to increase cold-adapted amylase production using two statistical experimental methods including Plackett–Burman design, which was applied to find the key ingredients for the best medium composition, and response surface methodology, which was used to determine the optimal concentrations of these components. The results showed starch, tryptone, and initial pH had significant effects on the cold-adapted amylase production. A central composite design was then employed to further optimize these three factors. The experimental results indicated that the optimized composition of medium was 6.38 g L⁻¹ starch, 33.84 g L⁻¹ tryptone, 3.00 g L⁻¹ yeast extract, 30 g L⁻¹ NaCl, 0.60 g L⁻¹ MgSO₄ and 0.56 g L⁻¹ CaCl₂. The optimized cultivation conditions for amylase production were pH 7.18, a temperature of 20°C, and a shaking speed of 180 rpm. Under the proposed optimized conditions, the amylase experimental yield (676.63 U mL⁻¹) closely matched the yield (685.60 U mL⁻¹) predicted by the statistical model. The optimization of the medium contributed to tenfold higher amylase production than that of the control in shake-flask experiments.     

Bioreduction of Cu(II) by Cell-Free Copper Reductase from a Copper Resistant <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NA

Environmental copper contamination is a serious human health problem. Copper reductase is produced by microorganisms to facilitate copper uptake by ATPases into the cells increasing copper biosorption. This study assessed the reduction of Cu(II) by cell-free extracts of a highly copper-resistant bacterium, Pseudomonas sp. strain NA, isolated from vineyard soil contaminated with copper. Both intact cells and cell-free extract of Pseudomonas sp. strain NA displayed substantial reduction of Cu(II). Intact cells reduced more then 80 mg L⁻¹ of Cu(II) from medium amended with 200 mg L⁻¹ of copper after 24 h of incubation. Cell-free extract of the isolate reduced more than 65% of the Cu(II) at initial copper concentration of 200 mg L⁻¹ after 24 h. Soluble protein production was high at 72 h of incubation at 100 mg L⁻¹ of copper, with more then 60 μg L⁻¹ of total soluble protein in cell-free extract recorded. Cu(II) reduction by isolate NA was increased when copper concentration increased for both intact cells and cell-free extract. Results indicate that Pseudomonas sp. strain NA produces copper reductase enzyme as the key mechanism of copper biotransformation.     

Effects of nitrogen sources on cell growth and lipid accumulation of green alga <Emphasis Type="Italic">Neochloris oleoabundans</Emphasis>

Microalgal lipids are the oils of future for sustainable biodiesel production. However, relatively high production costs due to low lipid productivity have been one of the major obstacles impeding their commercial production. We studied the effects of nitrogen sources and their concentrations on cell growth and lipid accumulation of Neochloris oleoabundans, one of the most promising oil-rich microalgal species. While the highest lipid cell content of 0.40 g/g was obtained at the lowest sodium nitrate concentration (3 mM), a remarkable lipid productivity of 0.133 g l⁻¹ day⁻¹ was achieved at 5 mM with a lipid cell content of 0.34 g/g and a biomass productivity of 0.40 g l⁻¹ day⁻¹. The highest biomass productivity was obtained at 10 mM sodium nitrate, with a biomass concentration of 3.2 g/l and a biomass productivity of 0.63 g l⁻¹ day⁻¹. It was observed that cell growth continued after the exhaustion of external nitrogen pool, hypothetically supported by the consumption of intracellular nitrogen pools such as chlorophyll molecules. The relationship among nitrate depletion, cell growth, lipid cell content, and cell chlorophyll content are discussed.     

Utilization of <Emphasis Type="Italic">Anabaena</Emphasis> sp. in CO<Subscript>2</Subscript> removal processes

This paper focuses on modelling the growth rate and exopolysaccharides production of Anabaena sp. ATCC 33047, to be used in carbon dioxide removal and biofuels production. For this, the influence of dilution rate, irradiance and aeration rate on the biomass and exopolysaccharides productivity, as well as on the CO₂ fixation rate, have been studied. The productivity of the cultures was maximum at the highest irradiance and dilution rate assayed, resulting to 0.5 g_bio l⁻¹ day⁻¹ and 0.2 g_eps l⁻¹ day⁻¹, and the CO₂ fixation rate measured was 1.0 gCO₂ l⁻¹ day⁻¹. The results showed that although Anabaena sp. was partially photo-inhibited at irradiances higher than 1,300 μE m⁻² s⁻¹, its growth rate increases hyperbolically with the average irradiance inside the culture, and so does the specific exopolysaccharides production rate. The latter, on the other hand, decreases under high external irradiances, indicating that the exopolysaccharides metabolism hindered by photo-damage. Mathematical models that consider these phenomena have been proposed. Regarding aeration, the yield of the cultures decreased at rates over 0.5 v/v/min or when shear rates were higher than 60 s⁻¹, demonstrating the existence of thus existence of stress damage by aeration. The behaviour of the cultures has been verified outdoors in a pilot-scale airlift tubular photobioreactor. From this study it is concluded that Anabaena sp. is highly recommended to transform CO₂ into valuable products as has been proved capable of metabolizing carbon dioxide at rates of 1.2 gCO₂ l⁻¹ day⁻¹ outdoors. The adequacy of the proposed equations is demonstrated, resulting to a useful tool in the design and operation of photobioreactors using this strain.     

Production of curdlan using sucrose or sugar cane molasses by two-step fed-batch cultivation of Agrobacterium species

Maltose and sucrose were efficient carbon sources for the production of curdlan by a strain of Agrobacterium sp. A two-step, fed-batch operation was designed in which biomass was first produced, followed by curdlan production which was stimulated by nitrogen limitation. There exists an optimal timing for nitrogen limitation for curdlan production in the two-step, fed-batch operation. Maximum curdlan production (60 g L⁻¹) was obtained from sucrose with a productivity of 0.2 g L⁻¹ h⁻¹ when nitrogen was limited at a cell concentration of 16.0 g L⁻¹. It was also noted that the curdlan yield from sucrose was as high as 0.45 g curdlan g⁻¹ sucrose, and the highest specific production rate was 1.0 g curdlan g⁻¹ cells h⁻¹ right after nitrogen limitation. Of particular importance was the use of molasses as a cheap carbon source to produce curdlan in the two-step, fed-batch cultivation. As high as 42 g L⁻¹ of curdlan with a yield of 0.35 g curdlan g⁻¹ total sugar was obtained after 120 h of fed-batch cultivation. Received 20 August 1996/ Accepted in revised form 26 November 1996     

Simultaneous nutrients and carbon removal during pretreated swine slurry degradation in a tubular biofilm photobioreactor

The biodegradation potential of an innovative enclosed tubular biofilm photobioreactor inoculated with a Chlorella sorokiniana strain and an acclimated activated sludge consortium was evaluated under continuous illumination and increasing pretreated (centrifuged) swine slurry loading rates. This photobioreactor configuration provided simultaneous and efficient carbon, nitrogen, and phosphorous treatment in a single-stage process at sustained nitrogen and phosphorous removals efficiencies ranging from 94% to 100% and 70–90%, respectively. Maximum total organic carbon (TOC), NH₄ ⁺, and PO₄ ³⁻ removal rates of 80 ± 5 g C m_r ⁻³ day⁻¹, 89 ± 5 g N m_r ⁻³ day⁻¹, and 13 ± 3 g P m_r ⁻³ day⁻¹, respectively, were recorded at the highest swine slurry loadings (TOC of 1,247 ± 62 mg L⁻¹, N–NH₄ ⁺ of 656 ± 37 mg L⁻¹, P–PO₄ ³⁺ of 117 ± 19 mg L⁻¹, and 7 days of hydraulic retention time). The unusual substrates diffusional pathways established within the phototrophic biofilm (photosynthetic O₂ and TOC/NH₄ ⁺ diffusing from opposite sides of the biofilm) allowed both the occurrence of a simultaneous denitrification/nitrification process at the highest swine slurry loading rate and the protection of microalgae from any potential inhibitory effect mediated by the combination of high pH and high NH₃ concentrations. In addition, this biofilm-based photobioreactor supported efficient biomass retention (>92% of the biomass generated during the pretreated swine slurry biodegradation).     

Kinetics of CO conversion into H<Subscript>2</Subscript> by <Emphasis Type="Italic">Carboxydothermus hydrogenoformans</Emphasis>

The objective of this study was to improve the biological water–gas shift reaction for producing hydrogen (H₂) by conversion of carbon monoxide (CO) using an anaerobic thermophilic pure strain, Carboxydothermus hydrogenoformans. Specific hydrogen production rates and yields were investigated at initial biomass densities varying from 5 to 20 mg volatile suspended solid (VSS) L⁻¹. Results showed that the gas–liquid mass transfer limits the CO conversion rate at high biomass concentrations. At 100-rpm agitation and at CO partial pressure of 1 atm, the optimal substrate/biomass ratio must exceed 5 mol CO g⁻¹ biomass VSS in order to avoid gas–liquid substrate transfer limitation. An average H₂ yield of 94 ± 3% and a specific hydrogen production rate of ca. 3 mol g⁻¹ VSS day⁻¹ were obtained at initial biomass densities between 5 and 8 mg VSS⁻¹. In addition, CO bioconversion kinetics was assessed at CO partial pressure from 0.16 to 2 atm, corresponding to a dissolved CO concentration at 70°C from 0.09 to 1.1 mM. Specific bioactivity was maximal at 3.5 mol CO g⁻¹ VSS day⁻¹ for a dissolved CO concentration of 0.55 mM in the culture. This optimal concentration is higher than with most other hydrogenogenic carboxydotrophic species.     

Optimization of culture conditions for tissue culture production of young plantlets of carrageenophyte <Emphasis Type="Italic">Kappaphycus</Emphasis>

To improve the production of Kappaphycus plantlets in tissue culture, optimum media concentrations of an Ascophyllum nodosum extract (Acadian Marine Plant Extract Powder, AMPEP), plant growth regulators (PGR), pH–temperature combinations, and explant density were determined. Kappaphycus alvarezii var. tambalang purple (PUR), kapilaran brown (KAP), vanguard brown (VAN), adik-adik (AA), tungawan green (TGR), and K. striatum var. sacol green (GS) were used as explants. Based on the shortest period for shoot emergence and the economical use of AMPEP, the optimum enriched media was 3.0 mg L⁻¹ AMPEP and 0.1 mg L⁻¹ AMPEP + PGR 1 mg L⁻¹ each phenylacetic acid (PAA) and zeatin for PUR, 1.0 mg L⁻¹ AMPEP + PGR for KAP and GS, 0.1 mg L⁻¹ AMPEP + PGR for VAN, and 3.0 mg L⁻¹ AMPEP and 0.001 mg L⁻¹ AMPEP + PGR for AA and TGR. Results showed that the addition of PGR to low concentrations of AMPEP hastened shoot formation. pH–temperature combinations for the most rapid shoot formation were determined for the brown (KAP) and purple (PUR) color morphotypes of K. alvarezii var. tambalang and the green morphotype of K. striatum var. sacol (GS) cultured in 1.0 mg L⁻¹ AMPEP + PGR. The brown morphotype produced the most number of shoots at pH 7.7 at 20°C after as little as 20 days. Purple K. alvarezii showed an increased shoot formation at pH 6.7 at 25°C and the green K. striatum morphotype at pH 8.7 at 25°C. The optimum number of explants added to the culture media was also determined for tungawan green (TGR), brown (KAP), and tambalang purple (PUR) varieties of K. alvarezii in 1.0 mg L⁻¹ AMPEP + PGR. The number of explants and the volume of the culture media combination were also tested. The highest average number of shoots formed occurred in two explants:1 mL culture media (2:1) for KAP and PUR (35.00% and 16.67%, respectively) and 1 explant: 2 mL culture media for the TGR (100.00%) with a range of 0.5–3.0 mm shoot length after 40 days in culture. The earliest shoot formation was observed after 21 days for the brown and 9 days for both the green and purple color morphotypes of Kappaphycus, in all densities investigated. This indicated that within the range tested, the density of explants did not have a significant effect on the rate of shoot formation but did influence the average number generated from the culture. The rate of production of new and improved Kappaphycus explants for a commercial nursery stock was improved through the use of AMPEP with optimized culture media pH, temperature, and density conditions.     

Ultrahigh-cell-density culture of a marine green alga Chlorococcum littorale in a flat-plate photobioreactor

To test the feasibility of CO₂ remediation by microalgal photosynthesis, a modified type of flat-plate photobioreactor [Hu et al. (1996) Biotechnol Bioeng 51:51–60] has been designed for cultivation of a high-CO₂-tolerant unicellular green alga Chlorococcum littorale. The modified reactor has a narrow light path in which intensive turbulent flow is provided by streaming compressed air through perforated tubing into the culture suspension. The length of the reactor light path was optimized for the productivity of biomass. The interrelationship between cell density and productivity, as affected by incident light intensity, was quantitatively assessed. Cellular ultrastructural and biochemical changes in response to ultrahigh cell density were investigated. The potential of biomass production under extremely high CO₂ concentrations was also evaluated. By growing C. littorale cells in this reactor, a CO₂ fixation rate of 16.7 g CO₂ l⁻¹ 24 h⁻¹ (or 200.4 g CO₂ m⁻² 24 h⁻¹) could readily be sustained at a light intensity of 2000 μmol m⁻² s⁻¹ at 25 °C, and an ultrahigh cell density of well over 80 g l⁻¹ could be maintained by daily replacing the culture medium. Received: 20 October 1997 / Received revision: 19 December 1997 / Accepted: 24 January 1998     

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch cultures of Pseudomonas sp EL-2

Pseudomonas sp EL-2 was cultivated to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from a structurally unrelated carbon source, glucose, by a fed-batch culture technique. Variation of the carbon to nitrogen (C/N) ratio of the medium produced optimal P(3HB-co-3HV) production at a C/N ratio of 95. Production of P(3HB-co-3HV) was favored by a dissolved oxygen tension of 40%. A maximum biomass concentration of 38 g L⁻¹ containing 53% P(3HB-co-3HV) was achieved after 45 h of cultivation. This corresponds to a volumetric productivity of 0.84 g L⁻¹ h⁻¹. The copolymer contained 7.5 mol% 3-hydroxyvalerate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 36–40. Received 28 January 1999/ Accepted in revised form 11 September 1999