Microalgal biomass production and on-site bioremediation of carbon dioxide, nitrogen oxide and sulfur dioxide from flue gas using Chlorella sp. cultures

The growth and on-site bioremediation potential of an isolated thermal- and CO?-tolerant mutant strain, Chlorella sp. MTF-7, were investigated. The Chlorella sp. MTF-7 cultures were directly aerated with the flue gas generated from coke oven of a steel plant. The biomass concentration, growth rate and lipid content of Chlorella sp. MTF-7 cultured in an outdoor 50-L photobioreactor for 6 days was 2.87 g L?1 (with an initial culture biomass concentration of 0.75 g L?1), 0.52 g L?1 d?1 and 25.2%, respectively. By the operation with intermittent flue gas aeration in a double-set photobioreactor system, average efficiency of CO? removal from the flue gas could reach to 60%, and NO and SO? removal efficiency was maintained at approximately 70% and 50%, respectively. Our results demonstrate that flue gas from coke oven could be directly introduced into Chlorella sp. MTF-7 cultures to potentially produce algal biomass and efficiently capture CO?, NO and SO? from flue gas.     

CO2 and O2 gas exchange in outdoor thin-layer high density microalgal cultures

Two thin layer culture units operated as batch cultures with the algaChlorella kessleri were used in gas exchange experiments. The mass transfer coefficient K_g [g m^–2 h^–1 kPa^–1] of O₂ and CO₂ desorption from culture surface decreased with increasing culture temperature. Between 60–70% of supplied CO₂ was used for algal growth. It was estimated that the length of growth surface may be extended to about 50 m, without additional saturation by CO₂. On average 1.35 g CO₂ was consumed by the alga per 1 g of produced O₂. Net CO₂ consumption (R_CO2) and O₂ production (R_O2) were not inhibited by irradiance. R_O2 did not decrease (in some cases it even increased) along the culture surface, despite increased accumulation of O₂. Measurement of pO₂ where the culture leaves the reactor before being pumped back onto the illuminated surface, correlated with O₂ production and CO₂ consumption and may be used to monitor the reactors growth performance.     

Utilisation of Chlorella vulgaris cell biomass for the production of enzymatic protein hydrolysates

Studies on enzymatic hydrolysis of cell proteins in green microalgae Chlorella vulgaris 87/1 are described. Different proteases can be used for production of hydrolysates from ethanol extracted algae. The influence of reaction parameters on hydrolysis of extracted biomass with pancreatin was considered, and the composition of hydrolysates (Cv-PH) was investigated in relation to the starting materials. Significant changes in the degree of hydrolysis were observed only during the first 2h and it remained constant throughout the process. An enzyme-substrate ratio of 30-45 units/g algae, an algae concentration of 10-15% and pH values of 7.5-8.0 could be recommended. Differences in the chromatographic patterns of Cv-PH and a hot-extract from Chlorella biomass were observed. Adequate amounts of essential amino acids (44.7%) in relation to the reference pattern of FAO for human nutrition were found, except for sulfur amino acids. Cv-PH could be considered as a potential ingredient in the food industry.     

Using chlorophyll fluorescence to monitor yields of microalgal production

A monitoring system for microalgal production was developed over a 12:12-h light:dark cycle at a steady state of growth to test the feasibility of estimating carbon production using the fluorescence method. An empirical linear relationship between the electron transport rate, based on the fluorescence method, and carbon assimilation, based on the conventional carbon method, was successfully obtained. The results demonstrate the relevance of the electron transport rate in determining carbon production of microalgae under steady-state growth conditions.     

A simple and low-cost airlift photobioreactor for microalgal mass culture

A simple, low-cost, and efficient airlift photobioreactor for microalgal mass culture was designed and developed. The reactor was made of Plexiglas, and composed of three major parts: outer tube, draft tube and air duct. The fluid-dynamic characteristics of the airlift reactor were studied. The system proved to be well suited to the mass cultivation of a marine microalga, Chlorella sp. In batch culture, the biomass volumetric output rate of 0.21 g l^–1 d^–1 was obtained at the superficial gas velocity of 4 mm s^–1 in the draft tube.     

On-line estimation of O<Subscript>2</Subscript> production,CO<Subscript>2</Subscript> uptake,and growth kinetics of microalgal cultures in a gas-tight photobioreactor

Growth of the green algae Chlamydomonas reinhardtii and Chlorella sp. in batch cultures was investigated in a novel gas-tight photobioreactor, in which CO₂, H₂, and N₂ were titrated into the gas phase to control medium pH, dissolved oxygen partial pressure, and headspace pressure, respectively. The exit gas from the reactor was circulated through a loop of tubing and re-introduced into the culture. CO₂ uptake was estimated from the addition of CO₂ as acidic titrant and O₂ evolution was estimated from titration by H₂, which was used to reduce O₂ over a Pd catalyst. The photosynthetic quotient, PQ, was estimated as the ratio between O₂ evolution and CO₂ up-take rates. NH₄ ⁺, NO₂ ⁻, or NO₃ ⁻ was the final cell density limiting nutrient. Cultures of both algae were, in general, characterised by a nitrogen sufficient growth phase followed by a nitrogen depleted phase in which starch was the major product. The estimated PQ values were dependent on the level of oxidation of the nitrogen source. The PQ was 1 with NH₄ ⁺ as the nitrogen source and 1.3 when NO₃ ⁻ was the nitrogen source. In cultures grown on all nitrogen sources, the PQ value approached 1 when the nitrogen source was depleted and starch synthesis became dominant, to further increase towards 1.3 over a period of 3–4 days. This latter increase in PQ, which was indicative of production of reduced compounds like lipids, correlated with a simultaneous increase in the degree of reduction of the biomass. When using the titrations of CO₂ and H₂ into the reactor headspace to estimate the up-take of CO₂, the production of O₂, and the PQ, the rate of biomass production could be followed, the stoichiometrical composition of the produced algal biomass could be estimated, and different growth phases could be identified.     

Alder-Frankia interaction and alder-poplar association for biomass production

Summary Alders have an important role to play in biomass producing stands because of their N₂-fixing ability and their capacity to withstand soils having an excess of moisture. The objectives of preliminary trials were (1) to find if there is any alder-genotype xFrankia-strain interaction when the effect of inoculating the bacteria was compared to no inoculation in seed beds of different species and provenances of alder, (2) to measure the possible effect of black alders interplanted in poplars compared to pure poplar plots. Two trials were laid out to study the alder-Frankia interaction. Both produced interaction. In the first one the inoculation had a favorable effect onAlnus glutinosa at age 2 years andA. cordata at age 1 and 2 and no effect onA. rubra. In the second one the inoculation had a depressive effect at age 1 on 2 of 3 provenances ofA. rubra and no effect on 1A. rubra, 3A.glutinosa and 3A. cordata provenances.A closely spaced field trial associating one black alder provenance and the poplar clone UNAL gives no superiority of mixed plots compared to pure plots. The results suggest that the N₂-fixation of alders is not profitable to poplars at age 3 with a 1.5×2 m spacing.     

Predominant growth of Alcanivorax during experiments on "oil spill bioremediation" in mesocosms

Mesocosm experiments were performed to study the changes on bacterial community composition following oil spill in marine environment. The analysis of 16S crDNA revealed a shift in the structure of initial bacterial population that was drastically different from that one measured after 15 days. The results showed that, after 15 days, bacteria closely related to the genus Alcanivorax became the dominant group of bacterial community in petroleum-contaminated sea water nitrogen and phosphorus amended. This suggested that these bacteria played the most important role in the process of bioremediation of oil-contaminated marine environments.     

Pseudomonas sp. to Sphingobium indicum: a journey of microbial degradation and bioremediation of Hexachlorocyclohexane

The unusual process of production of hexachlorocyclohexane (HCH) and extensive use of technical HCH and lindane has created a very serious problem of HCH contamination. While the use of technical HCH and lindane has been banned all over the world, India still continues producing lindane. Bacteria, especially Sphingomonads have been isolated that can degrade HCH isomers. Among all the bacterial strains isolated so far, Sphingobium indicum B90A that was isolated from HCH treated rhizosphere soil appears to have a better potential for HCH degradation. This conclusion is based on studies on the organization of lin genes and degradation ability of B90A. This strain perhaps can be used for HCH decontamination through bioaugmentation.     

Effects of nutrient amendments on fine root biomass in a primary successional forest in Hawai'i

Summary Thalli of Lobaria pulmonaria (L.) Hoffm., a nitrogen-fixing epiphyte common in mesic temperate forests, were collected in a Douglas-fir (Pseudotsuga menziesii Franco) forest near Corvallis, Oregon, and maintained for 20 to 40 days in controlled-environment chambers with atmospheric CO₂ concentrations of 374 and 700 ll^-1. Nitrogenase activity, which was assayed by the acetylene reduction method, was approximately doubled in the lichen maintained in elevated CO₂. Increases in nitrogen fixation by lichens may be an important part of the integrated ecosystem response to rising CO₂.     

Simultaneous and rapid monitoring of biomass and biopolymer production by Sphingomonas paucimobilis using Fourier transform-near infrared spectroscopy

The application of Fourier Transform near infrared spectroscopy (FT-NIRS) to near real-time monitoring of polysaccharide and biomass concentration was investigated using a gellan-producing strain of Sphingomonas paucimobilis grown in a stirred tank reactor. Successful models for both biomass and gellan were constructed despite the physichochemical complexity of the viscous process fluid. Modelling of biomass proved more challenging than for gellan, partly because of the low range of biomass concentration but a model with a good correlation coefficient (0.94) was formulated based on second derivative spectra. The gellan model was highly satisfactory, with an excellent correlation coefficient (0.98), again based on second derivative spectra. No sample pre-treatment was required and all spectral scanning was carried out on whole broth. Additionally, both models should be robust in practice since both were formulated using low numbers of factors. Thus, the near real time simultaneous monitoring of gellan and biomass in this highly complex matrix using FT-NIRS potentially opens the way to greatly improved process control strategies.     

A culture method for microalgal forms using two-tier vessel providing carbon-dioxide environment: studies on growth and carotenoid production

A culture method was developed for photoautotrophic culture of Haematococcus pluvialis, Chlorella vulgaris, Scenedesmus obliquus, Spirulina platensis, Nostoc and Stigonema in a two-tier flask consisting of nutrient media in the upper chamber and CO₂ generating buffer mixture (KHCO₃/K₂CO₃) in the lower chamber. The concentration of buffer mixture was varied to obtain desired levels of CO₂. CO₂ at 2.0% (v/v) level enhanced growth and chlorophyll content over control cultures (without CO₂ supplementation) in all microalgal species. Haematococcus pluvialis culture in BBM and KM1 media showed 6.71- and 2.07-fold increase in biomass yields with astaxanthin productivity at 7.26 and 7.48 mg l^–1 level respectively. CO₂ supplementation to C. vulgaris and S. obliquus cultures resulted in 5.97- and 7.30-folds increase in biomass with 2–3 fold increase in chlorophyll and carotenoid contents over their respective controls. Similarly 2–3 fold increase in chlorophyll and carotenoid contents were observed in Sp. platensis, Nostoc and Stigonema spp. This culture methodology will provide information on CO₂ requirement for growth of algae and metabolite production and also facilitates studies on the influence of light and temperature conditions.     

Evaluation of Gracilaria caudata J. Agardh for bioremediation of nutrients from shrimp farming wastewater

The accelerated development of shrimp farming in Brazil in recent decades has caused negative impacts to the environment. The most evident effects resulting from this activity is the increase in organic material, the reduction in oxygen and the excessive rise in water nutrients. Thus, there is a need for finding alternative solutions that can mitigate the negative impacts caused by this activity. A potentially viable solution is the use of macroalgae to remove nutrients from the cultivation systems. This study examined in situ (shrimp pond), the growth and storage of nitrogen and phosphorous from the macroalga Gracilaria caudata. A short-term measurement experiment was also conducted to evaluate the bioremediation potential this species. These results showed positive values for biomass and growth during the study period, except at day 45 for the tubular nets and day 75 for the cages, when they reached lower values than those of the initial weight. The results obtained indicate that G. caudata may reach annual production of 59.16 ton ha⁻¹ of wet weight, which corresponds to 11.83 ton dry weight. Nitrogen and phosphorous content in the algal tissues increased with time. The mean for the period was 2.61 ± 0.26% and 0.20 ± 0.03% for the nitrogen and phosphorous, respectively. An estimate of the data showed that 1 ha of cultivated algae has the potential to remove 0.309 ton ha⁻¹ year⁻¹ of nitrogen and 0.024 ton ha⁻¹ year⁻¹ of phosphorous. The study of the biofiltration capacity of G. caudata showed a significant reduction in nutrients. The removal of NH₄–N was around 59.5%, NO₃–N 49.6% and PO₄–P 12.3% in 4 h. These results suggest that although G. caudata showed relatively modest growth rates, they can be cultivated together with shrimp and can contribute to the removal of nitrogen and phosphorous from the pond. Moreover, the capacity to efficiently remove nutrients demonstrated in laboratory experiments encourages the use of this alga as a bioremediation agent.     

Meristem activity and biomass production as response mechanisms in two forest herbs

Actaea spicata and Geranium sylvaticum are deciduous rosette herbs, frequently coexisting in nutrient-rich forests of boreal Sweden. A. spicata is restricted to this habitat whereas G. sylvaticum has a wider ecological amplitude. To explore the different distributions of these species we studied the growth mechanisms by which they responded to nutrient and light supply. Plants raised from seeds were grown under four combinations of light and nutrient supplies and growth was followed for 20 weeks. Growth responses were measured as biomass accumulation and meristem activity, i.e. leaf development by shoot meristems. Biomass accumulation and meristem activity were affected by nutrient and light treatments. However, A. spicata responded more to nutrients, whereas G. sylvaticum responded more to light, and low resource supply shortened the time during which shoot meristems of A. spicata developed leaves without a corresponding effect on biomass accumulation. In G. sylvaticum, the periods of leaf development and biomass accumulation were the same, but a relationship between high development rates and peak allocation of biomass to shoot meristems indicated a storage function of these during leaf development. We conclude that the inflexible growth of A. spicata, mainly affected by nutrient supply, makes it less competitive in open habitats and restricts it to shaded, nutrient-rich habitats. G. sylvaticum should be more successful in habitats subjected to unpredictable disturbances, due to a flexible growth morphology and a dynamic function of meristems that buffers against variation in nutrient supply and facilitates positive light responses. Meristematic data should be included in interspecific comparisons on growth responses to different resource supplies. Unless species have the same growth mechanism, it is not satisfactory to study biomass-related traits alone, as internal constraints concerning plant development might confuse the interpretation.     

Hydrolysis of Chlorella biomass for fermentable sugars in the presence of HCl and MgCl2

When Chlorella biomass was hydrolyzed in the presence of 2% HCl and 2.5% MgCl₂, a sugar concentration of nearly 12%, and a sugar recovery of about 83% was obtained. Fermentation experiments demonstrated that glucose in the Chlorella biomass hydrolysates was converted into ethanol by Saccharomyces cerevisiae with a yield of 0.47 g g⁻¹, which is 91% of the theoretical yield. This chemical hydrolysis approach is thus a novel route for the hydrolysis of biomass to generate fermentable sugars.     

Heterotrophic production of ascorbic acid by microalgae

An aerobic fermentation process has been developed for the production of L-ascorbic acid (vitamin C). After an extensive screening program for microorganisms capable of heterotrophically synthesizing L-ascorbic acid, a unicellular green microalga,Chlorella pyrenoidosa, was selected. This organism has a number of characteristics that recommend it as an industrial organism: (1) it can double every 3.5 h when growing aerobically in the dark on a glucose-minimal salts medium; (2) its small size and tough cell wall make it very insensitive to shear, allowing very high impeller velocities; (3) it can be grown to 100 g L^–1 cell dry weight; (4) it is readily mutable by classical mutagenesis techniques; and (5) it has efficient growth kinetics with respect to yield of cell mass on glucose and oxygen. Fermentation process development and classical strain improvement have resulted in a greater than 70-fold increase in intracellular ascorbic acid concentration compared to the parent strainC. pyrenoidosa UTEX 1663. The process is compatible with existing industrial fermentation technology and equipment and is described in U.S. Patent 5,001,059. Patents have been submitted for a process in which the ascorbic acid accumulates extracellularly.     

Regulation of assimilatory nitrate reduction at the level of nitrite in Chlorella fusca

Batch cultures of Chlorella fusca excreted nitrite into the medium if gassed with air (0.03% CO₂), but they did not if supplied with air containing 5% CO₂. After a change from high to low CO₂ concentration in the gas stream, nitrite excretion started immediately. After an increase in CO₂ concentration to 5%, nitrite uptake started within only 30 min. Changes of in-vitro activities of nitrate reductase, nitrite reductase and glutamine synthetase did not correspond to changes of nitrite concentration in the medium and therefore could not explain these observations. A nitrite-binding site, whose activity corresponded with both nitrite excretion and uptake, was detected at the chloroplast envelope. From these data an additional regulatory step in the assimilatory nitrate-reduction sequence is suggested. This includes an envelopeprotein fraction probably regulating the availability of nitrite within the chloroplast.Abbreviations FMN riboflavin 5-phosphate - GS glutamine synthetase - NIR nitrite reductase - NR nitrate reductase     

Antibiotics-free stable polyhydroxyalkanoate (PHA) production from carbon dioxide by recombinant cyanobacteria

A practical antibiotics-free plasmid expression system in cyanobacteria was developed by using the complementation of cyanobacterial recA null mutation with the EscherichiacolirecA gene on the plasmid. This system was applied to the production of polyhydroxyalkanoate (PHA), a biodegradable plastic, and the transgenic cyanobacteria stably maintained the pha genes for PHA production in the antibiotics-free medium, and accumulated up to 52% cell dry weight of PHA.     

Upper limits of temperature for growth inChlorella (Chlorophyceae)

The upper limit of temperature for growth is a species-specific character in the genusChlorella. The limits of 14Chlorella species range from 26–30°C (C. saccharophila) to 38–42°C (C. sorokiniana), withC. fusca var.vacuolata (34°C) andC. kessleri (34–36°C) assuming an intermediate position. Thus, there is no wide gap in the temperature limits between the normal (“low-temperature”) species ofChlorella and the “high-temperature” species,C. sorokiniana.     

Simultaneous gas exchange and fluorescence measurements indicate differences in the response of sunflower,bean and maize to water stress

Gas exchange and fluorescence measurements of attached leaves of water stressed bean, sunflower and maize plants were carried out at two light intensities (250 mol quanta m^-2s^-1 and 850 mol quanta m^-2s^-1). Besides the restriction of transpiration and CO₂ uptake, the dissipation of excess light energy was clearly reflected in the light and dark reactions of photosynthesis under stress conditions. Bean and maize plants preferentially use non-photochemical quenching for light energy dissipation. In sunflower plants, excess light energy gave rise to photochemical quenching. Autoradiography of leaves after photosynthesis in ¹⁴CO₂ demonstrated the occurrence of leaf patchiness in sunflower and maize but not in bean. The contribution of CO₂ recycling within the leaves to energy dissipation was investigated by studies in 2.5% oxygen to suppress photorespiration. The participation of different energy dissipating mechanisms to quanta comsumption on agriculturally relevant species is discussed.Abbreviations F_o minimal fluorescence - F_m maximal fluorescence - F_p peak fluorescence - g leaf conductance - P_N net CO₂ uptake - q_N coefficient of non-photochemical quenching - q_P coefficient of photochemical quenching