Sophorolipid Production with High Yields on Whey Concentrate and Rapeseed Oil without Consumption of Lactose

Sophorolipids were produced by single-step batch cultivation of Candida bombicola ATCC 22214 on deproteinized whey concentrate and repeated feed of rapeseed oil. A mild sterilization method for whey was developed. High yields of 280 g dry sophorolipids l^–1 were obtained from deproteinized whey concentrate containing 100 g lactose l^–1 and 300 g rapeseed oil l^–1. Surprisingly, the whey lactose was not consumed by the organism. Growth only on the oil was assumed and a high lipase activity of 24 U per g cell dry weight resulted.     

Microbial production of single-cell protein from deproteinized whey concentrates

Deproteinized sweet and sour cheese whey concentrates were investigated for their suitability as substrates for the production of single-cell protein with Kluyveromyces marxianus CBS 6556 up to a 100-l scale. An important factor for gaining high cell concentrations was the use of the Crabtree-negative strain K. marxianus CBS 6556. Supplements such as trace elements, ammonium and calcium were required for the complete conversion of sweet whey concentrates into biomass, whereas sour whey concentrates had to be supplemented with ammonium, trace elements and vitamins. After improvement, biomass dry concentrations of up to 50 g l⁻¹ could be reached with Yx/s values of 0.52 for sweet whey and of up to 65 g l⁻¹ with Yx/s values of 0.48 for sour whey concentrates. The chemical oxygen demand of the whey concentrates were reduced by 80%. The cells were used for the analysis of amino acid and ash composition, showing a distinct increase of eight out of ten essential amino acids compared to sweet and sour whey protein and exceeding the World Health Organisation guidelines for valine, leucine, isoleucine, threonine, phenylalanine and tyrosine.     

Pullulan production from deproteinized whey by Aureobasidium pullulans

Aureobasidium pullulans P56 was investigated using an adaptation technique and a mixed culture system. The adaptation of A. pullulans and the mixed cultures of A. pullulans and/or Lactobacillus brevisX20, Debaryomyces hansenii 194 and Aspergillus niger did not increase the production of polysaccharide. Enzymic hydrolysis of lactose in deproteinized whey gave a higher polysaccharide concentration and polysaccharide yield than acidic hydrolysed lactose. Maximum polysaccharide concentration (11.0 ± 0.5 g L⁻¹), biomass dry weight (10.5 ± 0.4 g L⁻¹), polysaccharide yield (47.2 ± 1.8%) and sugar utilization (93.2 ± 2.8%) were achieved using enzyme-hydrolysed whey (pH 6.5) containing 25 g L⁻¹ lactose and supplemented with K₂HPO₄ 0.5%, L-glutamic acid 1%, olive oil 2.5%, and Tween 80 0.5%. In this case the pullulan content of the crude polysaccharide was 40%. Received 16 December 1997/ Accepted in revised form 12 March 1999     

Production of sophorolipids from whey: development of a two-stage process with Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214 using deproteinized whey concentrates as substrates

In order to produce sophorolipids from whey, thereby lowering the lactose content and biological oxygen demand, a two-step batch cultivation process was developed including medium sterilization by filtration. In the first step, whey was sterilized by a combination of crossflow and sterile filtration. Because the sophorolipid-producing yeast Candida bombicola ATCC 22214 was not able to use lactose as a carbon source directly, the oleaginous yeast Cryptococcus curvatus ATCC 20509 was grown on deproteinized whey concentrates (DWC). With 1: 1 diluted DWC-20, lactose was consumed as the carbon source and biomass (24 g/l dry weight content) as well as single-cell oil (SCO, 10 g/l) were produced. The cultivation broth was disrupted with a glass bead mill and it served as medium for growth (29 g cell dry mass/l) and sophorolipid production (12 g/l) of the yeast C. bombicola. Received: 29 July 1998 / Received revision: 5 October 1998 / Accepted: 11 October 1998     

Optimization of fermentation conditions for ethanol production from whey

Summary Optimal conditions for ethanol production in 7% whey solutions by the yeast Candida pseudotropicalis ATCC 8619 included initial pH of 4.57 and 30°C. Complete fermentation of the available lactose took place without supplementary nutrients; additions of nitrogen or phosphorus salts, yeast extract or corn steep liquor resulted in increased yeast production and lower ethanol yields. A positive correlation was observed between increases in yeast inocula and lactose utilization and ethanol production rates; 8.35 g/l of ethanol was obtained within 22 h by using yeast inoculum of 13.9 g/l. No differences in fermentation rates or ethanol yields were observed when whole or deproteinized whey solutions were used. Concentrated whey permeates, obtained after removal of the valuable proteins from whey, can be effectively fermented for ethanol production.     

Production of single cell protein from rice polishings using Candida utilis

Microbial protein was produced from defatted rice polishings using Candida utilis in shake-flasks and a 14-l fermentor to optimize fermentation conditions before producing biomass in a 50-l fermentor. The organism supported maximum values of 0.224 h⁻¹, 0.94, 1.35, 1.75, 2.12 g l⁻¹ h⁻¹, 0.62 g cells g⁻¹ substrate utilized and 0.38 g g⁻¹ for specific growth rate, true protein productivity, crude protein productivity, cell mass productivity, substrate consumption rate, cell yield, crude protein yield, respectively in 50-l fermentor studies using optimized cultural conditions. Maximum values compared favourably or were superior to published data in literature. The biomass protein in the 50-l fermentor contained 22.3, 27.8, 19.2, 9.5, 38.12, 8.5 and 0.27% true protein, crude protein, crude fibre, ash, carbon, cellulose and RNA content, respectively. The dried biomass showed a gross metabolizable energy value of 2678 kcal kg⁻¹ and contained all essential and non-essential amino acids. Yeast biomass as animal feed may replace expensive feed ingredients currently being used in poultry feed and may improve the economics of feed produced in countries like Pakistan. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nitrogen sequestration capacity of two salt marshes from the Tagus estuary

The role of salt marshes as nitrogen sink is examined taking into consideration the seasonal variation of above and belowground biomass of Spartina martima and Halimione portulacoides in two marshes from Tagus estuary, Pancas and Corroios, and the degradation rates of belowground litter. Total nitrogen was determined in plant components, decomposing litter and sediment. Biomass was higher in Corroios, the saltier marsh, with 7190 g m⁻² y⁻¹ dw of S. maritima and 6593 g m⁻² y⁻¹ dw of H. portulacoides and the belowground component contributed to 96% and 90% of total biomass, respectively. In the other marsh, Pancas, belowground biomass contributed to 56% and 76% of total biomass for S. maritima and H. portulacoides, respectively. Litterbag experiment showed that between 25% and 50% of nitrogen is lost within the first month and remained relatively constant in the next four months. Slower decomposition is observed in sediments with higher nitrogen concentration (max. 0.7% N in the saltier marsh). Higher concentrations of N were found in the sediment upper layers. Considering the sediment-root system, most of the nitrogen is stored in the sediment compartment and only about 1–4% of the total N was found in the roots. Considering these results, Tagus salt marshes act as a sink for nitrogen.     

Production of mosquitocidal <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> by solid state fermentation using agricultural wastes

In this study, Bacillus sphaericus NRC 69 was grown in culture media, in which 12 agricultural wastes were tested as the main carbon, nitrogen and energy sources under solid state fermentation. Of the 12 tested agricultural by-products, wheat bran was the most efficient substrate for the production of B. sphaericus mosquitocidal toxins against larvae of Culex pipiens (LC₅₀ 1.2 ppm). Mixtures of tested agricultural wastes separately with wheat bran enhanced the produced toxicity several folds and decreased LC₅₀ between 3.7- and 50-fold in comparison with that of agricultural wastes without mixing. The toxicity of B. sphaericus grown in wheat bran/rice hull at 8/2 (g/g) and wheat bran/barley straw at 1/4 (g/g) showed the same toxicity as that in wheat bran medium (LC₅₀ decreased 17- and 16-fold, in comparison with that in rice hull or barely straw media, respectively). In wheat bran medium, the maximum toxicity of the tested organism obtained at 50% moisture content, inoculum size 84 × 10⁶ CFU/g wheat bran and incubation for 6 days at 30°C. Addition of cheese whey permeate at 10% to wheat bran medium enhanced the toxicity of B. sphaericus NRC 69 about 46%.     

Protein production from whey usingPenicillium cyclopium; growth parameters and cellular composition

Summary The growth parameters ofPenicillium cyclopium have been evaluated in a continuous culture system for the production of fungal protein from whey. Dilution rates varied from 0.05 to 0.20 h^–1 under constant conditions of temperature (28°C) and pH (3.5). The saturation coefficients in the Monod equation were 0.74 g l^–1 for lactose and 0.14 mg l^–1 for oxygen, respectively. For a wide range of dilution rates, the yield was 0.68 g g^–1 biomass per lactose and the maintenance coefficient 0.005 g g^–1 h^–1 lactose per biomass, respectively. The maximum biomass productivity achieved was 2 g l^–1 h^–1 biomass at dilution rates of 0.16–0.17 h^–1 with a lactose concentration of 20 g l^–1 in the feed. The crude protein and total nucleic acid contents increased with a dilution rate, crude protein content varied from 43% to 54% and total nucleic acids from 6 to 9% in the range of dilution rates from 0.05 to 0.2 h^–1, while the Lowry protein content was almost constant at approximately 37.5% of dry matter.Nomenclature (mg l^–1) C_o initial concentration of dissolved oxygen - (h^–1) D dilution rate - (mg l^–1) K₀₂ saturation coefficient for oxygen - (g l^–1) K_s saturation coefficient for substrate - (g g^–1 h^–1) lactose per biomass) m maintenance energy coefficient - (mM g^–1 h^–1O₂ per biomass) Q₀₂ specific oxygen uptake rate - (g l^–1) S residual substrate concentration at steady state - (g l^–1) S_o initial substrate concentration in feed - (min) t_1/2 time when C_o is equal to C_o/2 - (g l^–1) X biomass concentration - (g l^–1) X biomass concentration at steady state - (g g^–1 biomass per lactose) Y_G yield coefficient for cell growth - (g g^–1 biomass per lactose) Y_x/s overall yield coefficient - (h^–1) specific growth rate     

Poly-(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli

Recombinant Escherichia coli strain GCSC 6576, harboring a high-copy-number plasmid containing the Ralstonia eutropha genes for polyhydroxyalkanoate (PHA) synthesis and the E. coli ftsZ gene, was employed to produce poly-(3-hydroxybutyrate) (PHB) from whey. pH-stat fed-batch fermentation, using whey powder as the nutrient feed, produced cellular dry weight and PHB concentrations of 109 g l⁻¹ and 50 g l⁻¹ respectively in 47 h. When concentrated whey solution containing 210 g l⁻¹ lactose was used as the nutrient feed, cellular dry weight and PHB concentrations of 87 g l⁻¹ and 69 g l⁻¹ respectively could be obtained in 49 h by pH-stat fed-batch culture. The PHB content was as high as 80% of the cellular dry weight. These results suggest that cost-effective production of PHB is possible by fed-batch culture of recombinant E. coli using concentrated whey solution as a substrate. Received: 19 December 1997 / Received revision: 17 March 1998 / Accepted: 20 March 1998     

Continuous production ofl-lactic acid from whey permeate by immobilizedLactobacillus casei subsp.casei

Summary The production of l-lactic acid from whey permeate, a waste product of the dairy industry, by fermentation with the lactic acid bacterium Lactobacillus casei subsp. casei was investigated. A fermentation medium consisting of permeate and supplements, which enables exponential growth of the organisms, was developed. A fast method for determination of free and immobilized biomass in solid-rich media, based on measurement of cellular ATP, was evolved. Continuous fermentations in a stirred tank reactor (STR) and in a fluidized bed reactor (FBR) with immobilized biomass were compared. In the STR a volumetric productivity of 5.5 g/l per hour at 100% substrate conversion [dilution rate (D) = 0.22 h^–1] was determined. In the FBR porous sintered glass beads were used for immobilization and a maximum biomass concentration of 105 g/kg support was measured. A productivity of 10 g/l per hour was obtained at D = 0.4 h^–1 (substrate conversion 93%) and of 13.5 g/l per hour at D = 1.0 h^–1 (substrate conversion 50%). Offprint requests to: W. Krischke     

Distillery and curd whey wastes as viable alternative sources for biosurfactant production

Biosurfactant production from synthetic medium and industrial waste, viz. distillery and whey wastes was investigated by using an oily sludge isolate Pseudomonas aeruginosa strain BS2. In synthetic medium separately supplemented with glucose and hexadecane as water-soluble and -insoluble carbon sources, respectively, strain BS2 reduced the surface tension of the fermentation broth from 57 to 27 mN/m. The culture produced biosurfactant during the stationary growth phase and its yield was 0.97 g/l. The culture utilized distillery and whey wastes for its growth, as maximum cell counts reached to 54 × 10⁸ and 64 × 10⁹ c.f.u./ml from an initial inoculum size of 1 × 0⁵ c.f.u./ml, respectively, within 48 h of incubation and in these wastes the yields of biosurfactant obtained were 0.91 and 0.92 g/l, respectively. In synthetic medium, distillery and whey wastes, strain BS2 produced a crystalline biosurfactant which belonged to the category of secondary metabolites and its maximum production occurred after the onset of nitrogen-limiting conditions. After recovering biosurfactant from the fermented waste, the chemical oxygen demand (COD) of distillery and whey wastes was significantly reduced by 81 and 87%, respectively. Total acids, nitrogen and phosphate levels in distillery waste were reduced by 90, 92 and 92%, respectively, while in case of whey waste the concentration of these nutrients was reduced by 88, 95 and 93%, respectively. The isolated biosurfactant possessed potent surface active properties, as it effectively reduced the surface tension of water from 72 to 27 mN/m and formed 100% stable emulsions of a variety of water-insoluble compounds such as hydrocarbons, viz. hexadecane, crude oil, kerosene and oily sludge and pesticides, viz. dichlorodiphenyltrichloroethane (DDT) and benzene hexachloride (BHC). The effectiveness of biosurfactant was also evident from its low critical micellar concentration (CMC) which was 0.028 mg/ml.     

Propionic acid and biomass production using continuous ultrafiltration fermentation of whey

Summary This paper presents a study of propionic acid and propionibacteria production from whey by usingPropionibacterium acidi-propionici in continuous fermentation with cell recycle. The highest propionic acid volumetric productivity achieved was 5 g.l^–1.h^–1 with no biomass bleeding. A maximal biomass concentration of 130 g.l^–1 was achieved before initiating biomass bleeding to give a biomass volumetric productivity of 3.2 g.l^–1.h^–1 with a biomass of 75 g.l^–1 and a propionic acid productivity of 3.6 g.l^–1.h^–1 (for about 100 hours i.e. more than 50 residence times).     

氮添加对中亚热带杜鹃灌丛凋落物生产和叶分解的影响

凋落物的生产和分解是生态系统养分循环的重要过程,受到大气氮沉降的深刻影响。但目前相关研究主要集中于森林和草地生态系统,氮沉降对灌丛生态系统凋落物养分归还的影响规律尚不清楚。因此选择亚热带分布广泛的杜鹃灌丛为研究对象,进行了为期两年的模拟氮沉降试验。试验设置4个处理:对照(CK, 0 g m-2 a-1)、低氮(LN, 2 g m-2 a-1)、中氮(MN, 5 g m-2 a-1)和高氮(HN, 10 g m-2 a-1)。结果显示:CK、LN、MN和HN 4种处理下,群落年平均凋落物量分别为(1936.54±358.9)、(2541.89±112.5)、(2342.97±519.8)、(2087.22±391.8) kg/hm²,LN、MN和HN处理样地的凋落量分别比对照样地高出32.68%、21.16%和7.93%;凋落叶、花果、凋落枝和其他组分占总凋落量的比例分别为75.75%、15.09%、7.70%和1.45%,不同浓度氮处理下各组分的凋落量均高于对照样地;凋落物组分表现出明显的季节动态:凋落叶在10—11月份达到峰值,凋落枝在10月份达到峰值,花果凋落物则在5月份凋落量最高,不同氮处理下凋落物的季节动态基本一致;白檀凋落叶分解速率显著高于杜鹃,二者分解95%所需时间分别为5.08—11.11 a和7.69—17.65 a,施氮使白檀凋落叶分解周期比对照样地缩短18.18%—54.28%;凋落叶分解过程中,N元素表现为富集-释放模式,P元素表现为富集模式。研究表明,氮添加能够促进群落中白檀凋落叶分解及N、P元素的释放,说明施氮可以调节凋落叶养分释放模式,对灌丛生态系统的养分循环具有调控作用。     

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.     

Fed-batch fermentation for production of Kluyveromyces marxianusFII 510700 cultivated on a lactose-based medium

A strain of Kluyveromyces marxianus was grown in batch culture in lactose-based media at varying initial lactose concentrations (10–60 g L^–1) at 30°C, pH 5.0, dissolved oxygen concentrations greater than 20%. Increasing the concentration of mineral salts three-fold at 40 g L^–1 and 60 g L^–1 initial lactose concentration showed only a small increase in the yield of biomass, from 0.38 g g^–1 to 0.41 g g^–1, indicating that the initial batch cultures were not significantly nutrient- (mineral salts)-limited. A relatively high biomass concentration (105 g L^–1) was obtained in fed-batch culture following extended lactose feeding. An average specific growth rate (0.27 h^–1), biomass yield (0.38 g g^–1) and overall productivity (2.9 g L^–1 h^–1) were obtained for these fed-batch conditions. This fed-batch protocol provides a strategy for achieving relatively high concentrations and productivities of K. marxianus on other lactose-based substrate streams (e.g., whey) from the dairy industry.     

Southern elephant seal (Mirounga leonina) II. Studies of milk protein fractions by gel electrophoresis

 Milk protein fractions during various stages of lactation in the southern elephant seal Mirounga leonina were analysed. Twelve milk samples were taken from ten females throughout the lactation period during 1990 and 1991 at Stranger Point, King George Island, South Shetland Islands. Milk samples were subjected to polyacrylamide gel electrophoresis (PAGE). Samples from different days of lactation gave similar qualitative electrophoretic patterns. True protein content was significantly higher (P<0.05) at the beginning of lactation, and then remained constant until weaning. Caseins and whey proteins each consisted of several protein entities (four and five distinct bands respectively). Casein constituted only about 30% of the protein nitrogen, the remaining 70% being derived from whey proteins. There was some variation in concentration of casein and whey proteins as a function of time (P<0.0.5). Received: 28 July 1993/Accepted: 25 July 1995     

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.     

Nitrogen mineralization ofSesbania sesban used as green manure for lowland rice in Sri Lanka

Sesbania sesban was evaluated as green manure crop for lowland rice in the Dry Zone of Sri Lanka. The legume was grown during a fallow period before lowland rice (Oryza sativa) and ploughed under just before transplanting. Weight loss and nitrogen content in litterbags containing leaves, stems and roots of the legume were monitored. Comparisons were made between rice yields from 20 m² plots after green manuring in combination with different nitrogen fertilizer levels (0, 2.4, 4.8 and 7.2 gm⁻²) and nitrogen fertilizer (9.6 gm⁻²) alone. Above-ground biomass ofS. sesban was 440 gm⁻² (dry wt) when ploughed under after 84 days growth. N-content in leaves, stems and roots was 3.76%, 0.41% and 0.73%, respectively. This gave a N-input fromS. sesban of 9.2 gm⁻² (8.3 g from above-ground parts and 0.9 g from roots). The corresponding K and P inputs were 7.3 and 0.6 gm⁻² respectively. The nitrogen rich leaves, which contained 88% of the nitrogen in the above-ground parts, decomposed and released its nitrogen much more rapidly than the stems and roots. After only four days the leaves had released 5.3 g Nm⁻² and after 14 days they had released 6.4 g Nm⁻². The highest rice yield (505 gm⁻²) was obtained usingS. sesban and 4.8 gm⁻² of N-fertilizer. The yields with only N-fertilizer or onlyS. sesban were 442 gm⁻² and 396 gm⁻², respectively. Due to the rapid decomposition of the nitrogen rich leaves,S. sesban did not behave as a slow release fertilizer. Thus, it is not necessary to apply nitrogen fertilizers as a basal dose.     

Cultivation of Chlorella pyrenoidosa in soybean processing wastewater

Chlorella pyrenoidosa was cultivated in soybean processing wastewater (SPW) in batch and fed-batch cultures without a supply of additional nutrients. The alga was able to remove 77.8 ± 5.7%, 88.8 ± 1.0%, 89.1 ± 0.6% and 70.3 ± 11.4% of soluble chemical oxygen demand (SCOD_Cr), total nitrogen (TN), NH₄⁺-N and total phosphate (TP), respectively, after 120 h in fed-batch culture. C. pyrenoidosa attained an average biomass productivity of 0.64 g L⁻¹ d⁻¹, an average lipid content of 37.00 ± 9.34%, and a high lipid productivity of 0.40 g L⁻¹ d⁻¹. Therefore, cultivation of C. pyrenoidosa in SPW could yield cleaner water and useful biomass.