Membrane inlet—ion mobility spectrometry with automatic spectra evaluation as online monitoring tool for the process control of microalgae cultivation

The cultivation of algae either in open raceway ponds or in closed bioreactors could allow the renewable production of biomass for food, pharmaceutical, cosmetic, or chemical industries. Optimal cultivation conditions are however required to ensure that the production of these compounds is both efficient and economical. Therefore, high-frequency analytical measurements are required to allow timely process control and to detect possible disturbances during algae growth. Such analytical methods are only available to a limited extent. Therefore, we introduced a method for monitoring algae release volatile organic compounds (VOCs) in the headspace above a bioreactor in real time. This method is based on ion mobility spectrometry (IMS) in combination with a membrane inlet (MI). The unique feature of IMS is that complete spectra are detected in real time instead of sum signals. These spectral patterns produced in the ion mobility spectrum were evaluated automatically via principal component analysis (PCA). The detected peak patterns are characteristic for the respective algae culture; allow the assignment of the individual growth phases and reflect the influence of experimental parameters. These results allow for the first time a continuous monitoring of the algae cultivation and thus an early detection of possible disturbances in the biotechnological process.     

A closed system for outdoor cultivation of Porphyridium

Experience accumulated during the past few years indicates that the main problems in the large-scale cultivation of algae in open ponds are low productivity and contamination. Thus, the use of closed systems can be an alternative method of cultivation. In the present study, a closed system made of polyethylene sleeves was compared with open ponds with respect to growth and polysaccharide production of two species of Porphyridium: Porphyridium sp. and P. aerugineum. For both species, cell number, biomass, and polysaccharide production were higher in the sleeves than in the ponds. It seems that polyethylene sleeves have the following advantages over open ponds: high light availability, high rate of heating and cooling, improved turbulence, relative lack of contamination, and prevention of evaporation and hence of fluctuation in salinity.     

Herbicide-resistant mutants of Botryococcus braunii race B (strain BOT-22)

The freshwater green alga Botryococcus braunii produces long-chain hydrocarbon oil in large quantities and secretes these from the cells. To exploit B. braunii as a source of next-generation biofuel, development of cost-effective cultivation systems are required. One of the most cost-effective methods for large-scale production is to simply grow B. braunii in open ponds. However, trials to cultivate B. braunii in open ponds often fail due to thriving of other green algal and cyanobacterial species because of the relatively slow growth of B. braunii. We previously demonstrated that herbicides are effective in control of contaminating algae. In order to use herbicide-assisted cultivation systems, we generated herbicide-resistant mutants of an oil-rich strain of B. braunii race B (strain BOT-22) by ethyl methanesulfonate mutagenesis. We isolated 44 glufosinate-resistant and 21 methyl viologen-resistant mutant lines. Some of these mutant lines exhibited vigorous growth and oil production in herbicide-containing liquid media, suggesting that they can be directly used in herbicide-containing cultivation systems.     

对虾养殖池塘底质修复技术对原生动物群落结构的影响

在对虾养殖池塘水体中共检测到原生动物86种,其中纤毛虫48种;鞭毛虫25种,肉足虫13种.原生动物优势种主要由一些运动能力相对较弱或固着生长的种类组成,如褐砂壳虫(Difflugia avellana)、瓜形膜袋虫(Cyclidium citrullus)、透明鞘居虫(Vaginicola crystalline)、多态喇叭虫(Stentor polymorphrus)和钟虫(Vorticella sp.)等,这些原生动物有以腐质和藻类为食、耐污性较强的特点.结果表明:在养殖初期,使用底质修复技术的试验池塘原生动物群落结构恢复比对照塘快,PFU群集的原生动物种类数和密度比对照塘多,水质比对照塘好,嗜污性相对较强的一些纤毛虫如尾草履虫(Paramecium caudaium)、拟瘦尾虫(Paruroleptus spp.)等比对照塘出现晚.在随后的养殖过程中,使用底质修复技术的试验塘原生动物群落结构比对照塘稳定,原生动物种类数和密度起伏小,pH值、透明度变化幅度也小于对照池塘,没有发生病害,水质较对照塘好.     

Insight into the studies of the practical use of microalgae in the former Soviet Union

Microalgae have been intensively cultivated and used in the national economy of the Soviet Union in Russia (Moscow, Leningrad, Pushcino, Krasnoyarsk, Irkutsk), the Ukraine, Byelorussia (Minsk), Azerbaijan (Baku), Uzbekistan (Tashkent), Latvia (Riga) and other former Soviet republics. The first studies on the practical use of microalgae started already at the beginning of last century and were connected with treatment of waste water in biological ponds. Ecophysiological studies of marine plankton algae in mono- and mixed cultures as well as in experimental systems with zooplankton organisms started at the end of the 1950s. In the 1960s, started to apply different equipments for cultivation of microalgae in open water basins and closed ecological systems. Research innovations were introduced in agriculture. The productivity of aquatic ecosystems and the distribution of energy within food chains were studied. Intensive research on microalgae continued, paying attention to the chemical composition, physiological and biochemical features. Nutrient supply appeared as one of the main problem for productivity of algal cultures, along with the procession, biochemistry of nutrients and nutrition value of organic matter produced by algae. The most attention was paid to the Chroococcales proteins and possibilities for their use in man-made closed ecological systems. The potential use of microalgae for regeneration of the atmosphere and purification of water in spacecrafts was studied. During the 1970s attention was paid to the role of algae in self-purification processes and biodegradation of surface-active substances in water bodies. Research in microalgae as bioindicators for the assessment of environment quality of aquatic ecosystems and as test objects for biotesting of natural and waste waters was conducted from the 1970s to 1980s.     

Effects of tadpole grazing on periphytic algae in ponds

Tadpole impact on periphytic algae was estimated with an exclosure experiment in the field. Algae growth on control (exposed) and experimental (netted-in) Perspex plates was measured in 12 ponds in 1991 and 8 in 1993.An index of snail (Lymnea and Planorbis)presence in the ponds was used to correct their effect. Grazing pressure (difference in amount of algae between control and experimental plates)differed between ponds. The difference was correlated to amount of Rana tadpoles present in the pond. At the higher densities of tadpoles observed in the study ponds, the algae standing crop on exposed plates was about 50–75% of that on netted-in plates. These result demonstrate that there is potential for effects of exploitation competition in ponds with a high tadpole density. This revised version was published online in July 2006 with corrections to the Cover Date.     

Summer algal communities and primary productivity in fish ponds

The paper presents data on primary productivity and phytoplankton communities in new experimental ponds which received the following treatments; ammonium nitrate and triplesuperphosphate, triplesuperphosphate, cracked corn (10% crude protein) and Auburn No. 3 fish feed (36% crude protein). Comparative data on algal communities were also obtained from production ponds which received feeds or fertilizers. Basic ecological data on macro-algae are also presented.

1. All nutrient additions to experimental ponds resulted in higher levels of gross photosynthesis and greater concentrations of chlorophyll a than were found in the control treatments. Fertilization with both nitrogen and phosphorus gave the highest values. Chlorophyll a and gross photosynthesis were higher in ponds receiving high protein content feed (Auburn No. 3) than in ponds to which low protein content feed (corn) was applied.
2. Persistent blooms of blue-green algae occurred in ponds receiving nitrogen and phosphorus fertilization. Phosphorus only fertilization produced blooms of blue-greens, but these blooms did not persist as in the ponds to which nitrogen was also added. Control ponds were dominated by green algae. Blue-green algae were seldom abundant in feed treatments.
3. Production ponds had high level of gross photosynthesis and large concentrations of chlorophyll a.
4. Many of the production ponds which received feed applications developed heavy blooms of blue-green algae.
5. The major species of blue-green algae observed in the present study were Oscillatoria sp., Raphidiopsis curvata, Anacystis nidulans, A. aeruginosa, Spirulina sp., and Anabaena circinalis. Heterocyst bearing forms, which can presumably fix nitrogen, were seldom noted in ponds that received continuous additions of nitrogen from fish feeds.
6. Macro-algae are abundant in many fish ponds. Data illustrating the competition of macro-algae with phytoplankton are presented.

     

Commercial production of microalgae: ponds, tanks, tubes and fermenters

The commercial culture of microalgae is now over 30 years old with the main microalgal species grown being Chlorella and Spirulina for health food, Dunaliella salina for β-carotene, Haematococcus pluvialis for astaxanthin and several species for aquaculture. The culture systems currently used to grow these algae are generally fairly unsophisticated. For example, Dunaliella salina is cultured in large (up to approx. 250 ha) shallow open-air ponds with no artificial mixing. Similarly, Chlorella and Spirulina also are grown outdoors in either paddle-wheel mixed ponds or circular ponds with a rotating mixing arm of up to about 1 ha in area per pond. The production of microalgae for aquaculture is generally on a much smaller scale, and in many cases is carried out indoors in 20–40 l carboys or in large plastic bags of up to approximately 1000 l in volume. More recently, a helical tubular photobioreactor system, the BIOCOIL™, has been developed which allows these algae to be grown reliably outdoors at high cell densities in semi-continuous culture. Other closed photobioreactors such as flat panels are also being developed. The main problem facing the commercialisation of new microalgae and microalgal products is the need for closed culture systems and the fact that these are very capital intensive. The high cost of microalgal culture systems relates to the need for light and the relatively slow growth rate of the algae. Although this problem has been avoided in some instances by growing the algae heterotrophically, not all algae or algal products can be produced this way.     

自养和兼养条件下蛋白核小球藻昼夜节律的响应

【目的】研究自养和兼养两种培养方式对蛋白核小球藻(Chlorella pyrenoidosa)生长、细胞分裂和生化组分积累的影响,探讨人工培养蛋白核小球藻的昼夜节律响应机制和优化技术。【方法】小球藻自养培养采用BG11培养基,兼养培养基在BG11培养基中添加4种不同浓度(1、5、10、20 g/L)的葡萄糖,培养周期为10 d。血球板计数法测定藻细胞浓度,干重法测定藻细胞生物量。显微观察藻细胞大小和分裂情况。脂染色法测定小球藻总脂的含量,藻细胞的叶绿素、蛋白和淀粉分别采用甲醇、氢氧化钠、硝酸钙浸提后通过紫外分光光度法定量测定。【结果】葡萄糖兼养培养对蛋白核小球藻具有显著的促生长效应,最适浓度为10 g/L。10 d收获时,兼养组(10 g/L葡萄糖)藻细胞浓度和干重分别是自养组的2.57倍和6.73倍。分析一昼夜中的藻细胞增殖规律可知,第2天和第5天时自养组中增殖的新生子细胞约有76.00%在黑暗期分裂产生,而兼养组中第2天和第5天光照期的新细胞增殖量占比分别达到40.90%和67.50%。一昼夜内藻细胞大小的迁移动态监测表明,第2天自养组藻细胞的体积变化静息期为8 h,兼养组只有4 h;第5天两组藻细胞大小迁移动态的昼夜节律明显,但兼养组黑暗结束后较大细胞(D6μm)占比显著高于自养组。第8天时,兼养组藻细胞已处于稳定期,总脂和蛋白含量均显著高于自养组,藻细胞总脂和色素含量在一昼夜中相对稳定,但蛋白和淀粉含量分别在光照8 h和12 h左右达到峰值。从第2天开始,对兼养组细胞每天进行2 h光延长,收获时藻细胞浓度和干重分别比对照组提高13%和11%。【结论】葡萄糖兼养培养能大幅提高蛋白核小球藻的生物量。蛋白核小球藻生长增殖与生化组分积累均受昼夜节律调控,自养条件下藻细胞以光照期生长黑暗期增殖为主。兼养培养提高藻细胞生物量的机制在于缩短藻细胞生长静息期,在昼夜节律中加速藻细胞生长并显著提高通过细胞周期检查点的细胞比例,光照期效应尤其明显。藻细胞蛋白和淀粉含量昼夜节律明显,最佳收获时间分别在光照8 h和12 h后。     

Algal biofuels

The world is facing energy crisis and environmental issues due to the depletion of fossil fuels and increasing CO₂ concentration in the atmosphere. Growing microalgae can contribute to practical solutions for these global problems because they can harvest solar energy and capture CO₂ by converting it into biofuel using photosynthesis. Microalgae are robust organisms capable of rapid growth under a variety of conditions including in open ponds or closed photobioreactors. Their reduced biomass compounds can be used as the feedstock for mass production of a variety of biofuels. As another advantage, their ability to accumulate or secrete biofuels can be controlled by changing their growth conditions or metabolic engineering. This review is aimed to highlight different forms of biofuels produced by microalgae and the approaches taken to improve their biofuel productivity. The costs for industrial-scale production of algal biofuels in open ponds or closed photobioreactors are analyzed. Different strategies for photoproduction of hydrogen by the hydrogenase enzyme of green algae are discussed. Algae are also good sources of biodiesel since some species can make large quantities of lipids as their biomass. The lipid contents for some of the best oil-producing strains of algae in optimized growth conditions are reviewed. The potential of microalgae for producing petroleum related chemicals or ready-make fuels such as bioethanol, triterpenic hydrocarbons, isobutyraldehyde, isobutanol, and isoprene from their biomass are also presented.     

虾池拟柱胞藻爆发的生态因子调查

2002～2003年,在广东珠三角地区凡纳滨对虾(Litopenaeus vannamei)低盐度养殖虾池水体生态的调查过程中发现部分虾池爆发拟柱胞藻(Cylindrospermopsis raciborskii).在调查的低盐度虾池中,共统计20个拟柱胞藻爆发的虾池,其种群密度平均高达7.74×10⁷ filaments·L^-1,平均优势度达47.0%.虾池水体平均水温为30.3℃,pH为8.9,盐度1.4,透明度19cm,化学耗氧量为24.3mg·L^-1.拟柱胞藻爆发的虾池水体环境表现为高水温、高pH、高有机质、低盐度且水体不断被搅动;通常爆发在对虾养殖的中后期富营养化水体.调查发现大量的拟柱胞藻对虾池其它藻类和微生物菌群表现一定的抑制作用,蓝纤维藻和小环藻等少数藻种可与拟柱胞藻形成共优势种.拟柱胞藻为广东珠三角凡纳滨对虾低盐度虾池重要优势藻种,且以螺旋型为主.     

江苏省灌西盐场盐藻及其β—胡萝卜素资源调查

为了开发利用富含β-胡萝卜素的江苏沿海盐田盐藻(Dunaliellasalina)资源,我们采用常规调查方法,对江苏省灌西盐场的天然盐藻进行调查,并推算出约有259公斤左右的β-胡萝卜素年产量。而且,藻数量和β-胡萝卜素含量都受到光照、温度、雨水等气候因素影响。本调查为灌西盐场盐藻资源开发提供了一个可靠的依据,为其它盐场估计本场资源量提供了一个参考。     

The effects of barley straw (Hordeum vulgare) on the growth of freshwater algae

Bioassays were conducted to determine the efficacy of barley straw liquor in controlling algal growth of 12 freshwater species of algae representing three divisions. Barley straw liquor inhibited the growth of three nuisance algae common in freshwater: Synura petersenii, Dinobyron sp., and Microcystis aeruginosa. However, Selenastrum capricornutum, Spirogyra sp., Oscillatoria lutea var. contorta, and Navicula sp. had significantly increased growth in the presence of straw liquor. The growth of the remainder, Ulothrix fimbriata, Scenedesmus quadricauda, Chlorella vulgaris, Anabaena flos-aquae, and Synedra sp. showed no significant difference from controls. In a related field study, we treated four of six ponds with barley straw and monitored their chlorophyll a levels for one growing season. While phytoplankton populations in all ponds decreased in midsummer, the phytoplankton biomass in treated ponds did not differ significantly from that of control ponds, suggesting that the application of barley straw had no effect on algal growth in these systems.     

Food habits, feeding behaviour and impact of triploid bighead carp, Hypophthalmichthys nobilis, in experimental ponds

Food of sterile triploid bighead carp, Hypophthalmichthys nobilis , was examined in ponds receiving water from a hypereutrophic take in Florida. No distinctive seasonal changes in food composition were found. The fish fed selectively on Botryococcus braunii Kuetzing, a large nuisance algal species, which constituted 61% of the volume and 50% of the dry weight of the food. Zooplankton was highly selected, but constituted only 3% of the food volume because of the low zooplankton density in the ponds. The remaining proportion of food consisted mainly of blue-green algae. Final fish biomass was low, ranging from 60 to 97 kg ha⁻¹ in individual ponds. Low biomass was due to slow fish growth and high fish mortality. Despite low biomass, the fish lowered the ratio of blue-green/green algae in the ponds and tended to lower phyto- and zooplankton abundance.     

光生物反应器培养微藻研究进展

微藻具有固定CO2和净化有机废水的能力,在环保、食品饲（饵）料、医药和生物能源开发等领域备受关注,但规模化培养及其产业化仍是研究的难点,亟待解决。就常用于大规模培养微藻的光生物反应器的特点和结构进行了综述。其中,封闭式微藻光生物反应器能够较好地调控藻种的培养条件、不易遭受污染,藻种的纯度容易控制,但培养规模小,生产成本较高;而开放式微藻光生物反应器无法精确控制藻种生长环境,但生产规模大、产量高、生产成本低,因此应用广泛。最佳的方法是综合两者优点,即首先利用封闭式微藻光生物反应器进行中试放大,大量繁殖藻种,然后投入开放式微藻光生物反应器内进行大规模商业生产,此方法有望成为微藻光生物反应器的发展方向,以期为微藻大规模培养提供参考借鉴。     

GROWTH DYNAMICS OF ULVA ROTUNDATA (CHLOROPHYTA) IN A FISH FARM: IMPLICATIONS FOR BIOMITIGATION AT A LARGE SCALE1

Changes in biomass of several macroalgae [Ulva rotundata Bliding; Gracilariopsis longissima (S. G. Gmel.) Steentoft, L. M. Irvine et Farnham; Ulva intestinalis L.; and Cladophora sp.] and marine plants (Zostera noltii and Ruppia cirrhosa) growing naturally in earthen ponds of a fish farm (Acuinova, San Fernando, Southern Spain) were recorded during a year. The farm is mainly devoted to the culture of gilthered seabream (Sparus aurata). The most conspicuous algal species thriving in the ponds was U. rotundata, which reached densities up to 600 g dry mass · m⁻² and produced up to 20.45 g C · m⁻² · d⁻¹. Dissolved nutrients (phosphate and ammonium), tissue nutrient content, and growth rates of this species were estimated during 2001 and 2002. Evidence of natural biomitigation by U. rotundata when water circulates throughout the fish farm is presented. Due to the fish cultivation, both phosphate and ammonium increased as water circulated from the preculture ponds to the postculture ponds. As a consequence, U. rotundata tissue nitrogen (N) and phosphorus (P) increased from algae growing in preculture ponds to algae growing in the outflow channel, so that mean C:N:P ratio varied from 773:57:1 in preculture ponds to 567:64:1 in the outflow channel. Phosphorus limited growth of U. rotundata during the spring. As growth rates increased as a function of tissue P, data were fitted to the Droop equation. From this equation, the estimated maximal growth rate was 0.295 ± 0.041 d⁻¹, the subsistence quota was 0.05 ± 0.01% P of dry mass, and the critical quota was 0.215% P of dry mass. The results suggest that management of the fish farm based on a large-scale integrated mariculture system of fish and macroalgae may increase the total ecological and economic benefits, both for the farm and for the environment.     

Use of an algal consortium of five algae in the treatment of landfill leachate using the high-rate algal pond system

Five species of microalgae Chlorella vulgaris, Scenedesmus quadricauda, Euglena gracilis, Ankistrodesmus convolutus and Chlorococcum oviforme, were screened for their ability to grow in treated landfill leachate (TL) using shake flask cultures. The treated leachate had undergone previous treatment through mechanical aeration in treatment ponds at the landfill site. The five algae, except for C. oviforme, were able to grow in medium containing up to 50% TL. Two high-rate algal ponds (HRAP) equipped with paddlewheel were used for the semi-continuous cultures. A mixture of the five algae was used to inoculate one of two HRAPs for secondary treatment of TL. The other HRAP was filled with natural lake water containing mixed populations of algae. A volume of 400?mL (1%) from both ponds were removed daily and replaced with TL. The leachate loading rate was increased to 2% (0.8?L?day^?1) on day?197 and then to 4% (1.6?L?day^?1) on day?309, providing hydraulic retention time of 100, 50 and 25?days, respectively. Although higher biomass was obtained in the HRAP containing the consortium of five algae, there was no significant difference in reduction of pollutants between the two ponds. The HRAPs produced algal biomass ranging from 2.00 to 5.54?g dry weight?L^?1 with significant reduction in chemical oxygen demand (91.0%), ammoniacal nitrogen (99.9%) and orthophosphate (86.0%) contents. The HRAP offers a potential treatment system for TL which is simple, low cost, flexible in use and requiring low maintenance.     

应用矿物盐改良池塘水质的研究

为了研究矿物盐对淡水养殖池塘水质的改良效果及应用前景,在12口土质池塘中对淡水养殖斑点叉尾鲴(Ictalurus punctatus Rafinesque,1818)施用三组矿物盐:NaNO3、KCl+MgCl2、NaNO3+KCl+MgCl3的对照实验,对照组池塘不施用任何矿物盐类;在182d的养殖期间,对所有处理组和对照组池塘的17项水质和5项底质因子进行了定期监测.结果表明,各处理组均显著降低了池塘水中的蓝藻相对密度;KCl+MgCl2的施用可促进水中硝化作用的进行;NaNO3+KCl+MgCl2的施用,显著提高了养殖池塘底泥表层的氧化还原电位;NaNO3+KCl+MgCl2组的池塘鱼产量比对照组显著提高6.6%,而饲料系数显著降低6.2%;NaNO3与KCl+MgCl2的共同施用起到了协同改良池塘水质和底质的作用.因此,NaNO3+KCl+MgCl2的应用可为淡水池塘水质的改良开辟一个新的途径,具有一定的应用前景.     

Obtaining plantlets from apical meristem of the red alga Gelidium sp.

For the first time, plantlets were obtained from fragments and cell aggregates (CA) of apical meristem of the red alga Gelidium sp. After two months of cultivation, an initial weight of 100 mg of fragments and CA from fresh meristem produced 3 g of plantlets without rhizoids. During the same period of cultivation, 100 mg of meristem fragments and CA isolated from thalli by the freezing-thawing procedure produced more than 20 g of plantlets with rhizoids. It is assumed that our methods for obtaining plantlets from fragments and CA of fresh and frozen-thawed meristem could be used to generate mass planting material for cultivation of algae (plantlets with rhizoids) in the sea and for tank-bubbling cultivation (plantlets without rhizoids). We speculate that meristem cells of frozen-thawed algae might be natural “seedstock” in the Arctic and Antarctic seas.     

链形藻类的中国新记录属—中眼藻属

中眼藻属(Mesostigma)被认为是与高等植物亲缘关系最近的单细胞鞭毛藻类, 是研究高等植物起源的关键类群之一。已有研究表明中眼藻主要分布于静止的淡水水体中, 诸如池塘、水库和小型湖泊等, 然而中国至今仍未有该藻的报道。本研究在重庆合川和福建九龙江采集到2株单细胞鞭毛藻类, 对其进行形态观察并基于18S rDNA序列进行了系统发育分析。研究结果表明这2株藻为中眼藻属的绿色中眼藻(Mesostigma viride)。此外, 研究从三峡库区的环境DNA中也鉴定出中眼藻的存在, 且主要出现在秋季, 说明秋季可能是在中国内陆采集中眼藻的最佳季节。研究是中眼藻属在中国的首次报道, 为今后的中眼藻相关研究奠定了基础。