Extraction of short chain chitooligosaccharides from fungal biomass and their use as promoters of arbuscular mycorrhizal symbiosis.

Short chain chitooligosaccharides (COs) are chitin derivative molecules involved in plant-fungus signaling during arbuscular mycorrhizal (AM) interactions. In host plants, COs activate a symbiotic signalling pathway that regulates AM-related gene expression. Furthermore, exogenous CO application was shown to promote AM establishment, with a major interest for agricultural applications of AM fungi as biofertilizers. Currently, the main source of commercial COs is from the shrimp processing industry, but purification costs and environmental concerns limit the convenience of this approach. In an attempt to find a low cost and low impact alternative, this work aimed to isolate, characterize and test the bioactivity of COs from selected strains of phylogenetically distant filamentous fungi: Pleurotus ostreatus, Cunninghamella bertholletiae and Trichoderma viride. Our optimized protocol successfully isolated short chain COs from lyophilized fungal biomass. Fungal COs were more acetylated and displayed a higher biological activity compared to shrimp-derived COs, a feature that-alongside low production costs-opens promising perspectives for the large scale use of COs in agriculture.

The CYPome of the model xenobiotic-biotransforming fungus Cunninghamella elegans.

The fungus Cunninghamella elegans is recognised as a microbial model of mammalian drug metabolism owing to its ability to catabolise xenobiotic compounds in an analogous fashion to animals. Its ability to produce phase I (oxidative) metabolites of drugs is associated with cytochrome P450 (CYP) activity; however, almost nothing is known about these enzymes in the fungus. In this paper we report the in silico analysis of the genome sequence of C. elegans B9769, which contains 32 genes putatively coding for CYPs. Based on their predicted amino acid sequences these were classified as belonging to CYP509, 5203, 5208, 5313, 5210, 61 and 51 families. Reverse transcription-quantitative PCR revealed that the gene coding for CYP5313D1 was significantly upregulated when C. elegans DSM1908 was cultivated in sabouraud dextrose in contrast to its expression in cells grown in Roswell Park Memorial Institute medium. This corresponded to the fungus' xenobiotic biotransformation ability when grown in the two media. Heterologous expression of cyp5313D1 in Pichia pastoris resulted in a recombinant strain that biotransformed flurbiprofen to 4'-hydroxyflurbiprofen, the same metabolite generated by C. elegans cultures. This is the first report of a xenobiotic-biotransforming CYP from this biotechnologically important fungus.

A Microbial Transformation Model for Simulating Mammal Metabolism of Artemisinin.

Artemisinin (ART) is a highly effective antimalarial agent isolated from the traditional Chinese herb Qinghao. Metabolism of ART and its derivatives in the body is one of the most pressing issues for pharmaceutical scientists. Herein, an efficient in vitro microorganism model for simulation of metabolism of ART in vivo was developed employing Cunninghamella elegans. Metabolites in the microbial transformation system and plasma of mice pre-administrated ART orally were analyzed by ultra-performance liquid chromatography (UPLC)-electrospray ionization (ESI)-quadrupole time-of-flight (Q-TOF)-mass spectrometry (MSE) combined with UNIFI software. Thirty-two metabolites were identified in vitro and 23 were identified in vivo. After comparison, 16 products were found to be common to both models including monohydroxylated ART, dihydroxylated ART, deoxyartemisinin, hydroxylated deoxyartemisinin, hydroxylated dihydroartemisinin (DHA), and hydroxylated deoxy-DHA. These results revealed that C. elegans CICC 40250 functioned as an appropriate model to mimic ART metabolism in vivo. Moreover, an overall description of metabolites of ART from C. elegans CICC 40250 has been provided. Notably, DHA was detected and identified as a metabolite of ART in mouse plasma for the first time.

[Lipid Composition in Cell Walls and in Mycelial and Spore Cells of Mycelial Fungi].

Qualitative and quantitative differences were found between the lipids of cell walls (CW) and of whole mycelial cells and dormant cells of mucoraceous and ascomycete fungi. Thus, whole mycelial cells (WC) contained more lipids than CW. Unlike sporangiospores and conidia (exogenous dormant spores), zygotes were found to have the highest content of triacylglycerol lipids (70%). Cell walls of mucoraceous fungi contained more triacylglycerols (TAG) and less polar lipids than ascomycete lipids. While all CW and WC studied were similar in fatty acid (FA) composition, their ratio was specific for each structure: linoleic acid predominated in mycelial CW and WC, while oleic acid was predominant in the spores; this difference was especially pronounced in conidial WC. Unlike WC, in CW massive lipids may be represented not by phosphatidylethanolamine (PEA) and phosphatidylcholine (PC), but by free fatty acids (FFA), free (FSt) and etherified sterols (ESt), phosphatidic acid (PA), fatty acid methyl esters (FAME), and glycolipids (GL), which is an indication of a special functional role of CW.

In situ hydrogen, acetone, butanol, ethanol and microdiesel production by Clostridium acetobutylicum ATCC 824 from oleaginous fungal biomass.

An in situ batch fermentation technique was employed for biohydrogen, acetone, butanol, ethanol and microdiesel production from oleaginous fungal biomass using the anaerobic fermentative bacterium Clostridium acetobutylicum ATCC 824. Oleaginous fungal Cunninghamella echinulata biomass which has ability to accumulate up to 71% cellular lipid was used as the substrate carbon source. The maximum cumulative hydrogen by C. acetobutylicum ATCC 824 from crude C. echinulata biomass was 260 ml H2 l(-1), hydrogen production efficiency was 0.32 mol H2 mole(-1) glucose and the hydrogen production rate was 5.2 ml H2 h(-1). Subsequently, the produced acids (acetic and butyric acids) during acidogenesis phase are re-utilized by ABE-producing clostridia and converted into acetone, butanol, and ethanol. The total ABE produced by C. acetobutylicum ATCC 824 during batch fermentation was 3.6 g l(-1) from crude fungal biomass including acetone (1.05 g l(-1)), butanol (2.19 g l(-1)) and ethanol (0.36 g l(-1)). C. acetobutylicum ATCC 824 has ability to produce lipolytic enzymes with a specific activity 5.59 U/mg protein to hydrolyze ester containing substrates. The lipolytic potential of C. acetobutylicum ATCC 824 was used as a biocatalyst for a lipase transesterification process using the produced ethanol from ABE fermentation for microdiesel production. The fatty acid ethyl esters (microdiesel) generated from the lipase transesterification of crude C. echinulata dry mass was analyzed by GC/MS as 15.4% of total FAEEs. The gross energy content of biohydrogen, acetone, butanol, ethanol and biodiesel generated through C. acetobutylicum fermentation from crude C. echinulata dry mass was 3113.14 kJ mol(-1). These results suggest a possibility of integrating biohydrogen, acetone, butanol and ethanol production technology by C. acetobutylicum with microdiesel production from crude C. echinulata dry mass and therefore improve the feasibility and commercialization of bioenergy production.

Biosurfactant-and-bioemulsifier produced by a promising Cunninghamella echinulata isolated from Caatinga soil in the northeast of Brazil.

A Mucoralean fungus was isolated from Caatinga soil of Pernambuco, Northeast of Brazil, and was identified as Cunninghamella echinulata by morphological, physiological, and biochemical tests. This strain was evaluated for biosurfactant/bioemulsifier production using soybean oil waste (SOW) and corn steep liquor (CSL) as substrates, added to basic saline solution, by measuring surface tension and emulsifier index and activity. The best results showed the surface water tension was reduced from 72 to 36 mN/m, and an emulsification index (E24) of 80% was obtained using engine oil and burnt engine oil, respectively. A new molecule of biosurfactant showed an anionic charge and a polymeric chemical composition consisting of lipids (40.0% w/w), carbohydrates (35.2% w/w) and protein (20.3% w/w). In addition, the biosurfactant solution (1%) demonstrated its ability for an oil displacement area (ODA) of 37.36 cm², which is quite similar to that for Triton X-100 (38.46 cm²). The stability of the reduction in the surface water tension as well as of the emulsifier index proved to be stable over a wide range of temperatures, in pH, and in salt concentration (4%-6% w/v). The biosurfactant showed an ability to reduce and increase the viscosity of hydrophobic substrates and their molecules, suggesting that it is a suitable candidate for mediated enhanced oil recovery. At the same time, these studies indicate that renewable, relatively inexpensive and easily available resources can be used for important biotechnological processes.

Succession patterns of fungi associated to wound-induced agarwood in wild Aquilaria malaccensis revealed from quantitative PCR assay.

Aquilaria malaccensis produces agarwood in response to wounding and fungal attack. However, information is limited regarding Aquilaria's interaction with its diverse fungal community. In this study, time-related changes of three natural fungal colonizers in two wounded wild A. malaccensis were tracked, beginning a few hours after wounding up to 12 months. Using species-specific primers derived from their nrITS sequences in quantitative real-time PCR (qPCR), we quantified the amount of Cunninghamella bainieri, Fusarium solani and Lasiodiplodia theobromae. Because time is a major factor affecting agarwood quantity and quality, 14 wood samples were collected at different time points, i.e., 0-18 h, 2-13 days, 2-18 weeks, and 6-12 months after wounding. qPCR data revealed that the abundance of the three species decreased over time. The fungi were detected in high numbers during the first few hours and days after wounding (40- to 25,000-fold higher levels compared with initial counts) and in low numbers (<1- to 3,200-fold higher than initially) many months later. Consistent with its role in defense response, the accumulation of secondary metabolites at the wounding site could have caused the decline in fungal abundance. Succession patterns of the two trees were not identical, indicating that fungal populations may have been affected by tree environment and wound microclimate. Our results are important for understanding the diversity of microbial community in wild Aquilaria species and their association to wound-induced agarwood formation. Fungi could be secondary triggers to agarwood production in situations where trees are wounded in attempt to induce agarwood.

Biotransformation of bavachinin by three fungal cell cultures.

Biotransformation of bavachinin (1) was investigated using three fungal cell cultures of Aspergillus flavus ATCC 30899, Cunninghamella elegans CICC 40250 and Penicillium raistrickii ATCC 10490, respectively. Two major converted products were identified by LC/MS, (1)H NMR and (13)C NMR and X-ray diffraction. Two biocatalyst systems, A. flavus ATCC 30899 and C. elegans CICC 40250 cell cultures, showed a great capacity of hydroxylation and two hydroxyl groups were attached at C-2″ and C-3″ positions in the side chain of the bavachinin A-ring, resulting in the formation of the same compound with a name, (S)-6-((R)-2,3-dihydroxy-3-methylbutyl)-2-(4-hydroxyphenyl)-7-methoxychromen-4-one (2). On the other hand, P. raistrickii ATCC 10490 cell cultures possessed the ability to reduction at C-4 of the substrate C-ring, resulting in the production of (2S,4R)-2-(4-hydroxyphenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)chromen-4-ol (3). Furthermore, the in vitro anti-tumor activities of the above compounds were evaluated by MTT assay. Compared with the substrate (1), product 3 possessed stronger inhibition activity on the human breast cancer cell line (MCF-7) and slightly lower inhibition activities against Hep G2, HeLa, Hep-2 and A549 cells lines; while the hydroxyl product 2 possessed much lower inhibition activity on tumor cells lines, which might be related to the insertion of two hydroxyl groups. Compounds 2 and 3 were considered to be novel. It was also the first time to biotransform bavachinin (1) by these three fungi, which suggested the potential role of microbial enzymes to synthesize novel compounds from plant secondary metabolites.

Optimization of aeration and agitation rate for lipid and gamma linolenic acid production by Cunninghamella bainieri 2A1 in submerged fermentation using response surface methodology.

The locally isolated filamentous fungus Cunninghamella bainieri 2A1 was cultivated in a 5 L bioreactor to produce lipid and gamma-linolenic acid (GLA). The optimization was carried out using response surface methodology based on a central composite design. A statistical model, second-order polynomial model, was adjusted to the experimental data to evaluate the effect of key operating variables, including aeration rate and agitation speed on lipid production. Process analysis showed that linear and quadratic effect of agitation intensity significantly influenced lipid production process (P < 0.01). The quadratic model also indicated that the interaction between aeration rate and agitation speed had a highly significant effect on lipid production (P < 0.01). Experimental results showed that a lipid content of 38.71% was produced in optimum conditions using an airflow rate and agitation speed of 0.32 vvm and 599 rpm, respectively. Similar results revealed that 0.058(g/g) gamma-linolenic acid was produced in optimum conditions where 1.0 vvm aeration rate and 441.45 rpm agitation rate were used. The regression model confirmed that aeration and agitation were of prime importance for optimum production of lipid in the bioreactor.

Enhancement of Cunninghamella elegans UCP/WFCC 0542 biomass and chitosan with amino acid supply.

Studies were carried out with Cunninghamella elegans UCP/WFCC 0542 to evaluate the effects of an abundant supply of amino acids, asparagine and corn steep liquor associated with sucrose on the production of biomass and chitosan by submerged fermentation. The concentrations of the components of the culture medium which were determined by a 2³ full factorial design evaluated the interactions and effects of the independent variables of the sucrose, asparagine and corn steep liquor in relation to carbon and nitrogen sources, on the production of chitosan regarding biomass. The best results were observed at the central point [asparagine 0.025%, sucrose 0.15% and 0.45% of corn steep liquor, ratio C:N=2:6], and produced maximum yields of 16.95 g/L biomass and 2.14 g/L chitosan, after 96 h of submerged fermentation. However, the lowest level of sucrose, asparagine and corn steep liquor produced a low amount of biomass (10.83 g/L) and chitosan (0.60 g/L). The infrared spectrum absorption of the chitosan produced by C. elegans showed bands regarding OH-axial stretching between 3406 and 3432 cm⁻¹, superimposed on the NH stretching band with axial deformation of the amide C=O group at about 1639 cm⁻¹, NH angular deformation at approximately 1560 cm⁻¹; axial deformation of amide-CN at around 1421 cm⁻¹, symmetrical angular deformation in CH3 at 1379 cm⁻¹, -CN axial deformation of amino groups from 1125 to 1250 cm⁻¹ and polysaccharide structure bands in the range of between 890-1150 cm⁻¹. The crystallinity index of chitosan was 60.92%, and its degree of deacetylation was 75.25%. A low percentage of a supply of sucrose and asparagine with corn steep liquor offered higher yields of biomass and chitosan production at low cost.

Cunninghamella echinulata causing fatally invasive fungal sinusitis.

We report a fatal case of invasive fungal sinusitis caused by Cunninghamella echinulata in a febrile, neutropenic 15-year-old male with relapsing acute leukemia. The isolate was recovered from a nasal biopsy from the right middle meatus, and microscopic examination of the tissue revealed angioinvasion and necrosis. Human infection caused by this organism has not been well documented; however, this report alerts us to its life-threatening potential.

Cadmium tolerance and removal from Cunninghamella elegans related to the polyphosphate metabolism.

The aim of the present work was to study the cadmium effects on growth, ultrastructure and polyphosphate metabolism, as well as to evaluate the metal removal and accumulation by Cunninghamella elegans (IFM 46109) growing in culture medium. The presence of cadmium reduced growth, and a longer lag phase was observed. However, the phosphate uptake from the culture medium increased 15% when compared to the control. Moreover, C. elegans removed 70%-81% of the cadmium added to the culture medium during its growth. The C. elegans mycelia showed a removal efficiency of 280 mg/g at a cadmium concentration of 22.10 mg/L, and the removal velocity of cadmium was 0.107 mg/h. Additionally, it was observed that cadmium induced vacuolization, the presence of electron dense deposits in vacuoles, cytoplasm and cell membranes, as well as the distinct behavior of polyphosphate fractions. The results obtained with C. elegans suggest that precipitation, vacuolization and polyphosphate fractions were associated to cadmium tolerance, and this species demonstrated a higher potential for bioremediation of heavy metals.

Filamentous fungal biofilm for production of human drug metabolites.

In drug development, access to drug metabolites is essential for assessment of toxicity and pharmacokinetic studies. Metabolites are usually acquired via chemical synthesis, although biological production is potentially more efficient with fewer waste management issues. A significant problem with the biological approach is the effective half-life of the biocatalyst, which can be resolved by immobilisation. The fungus Cunninghamella elegans is well established as a model of mammalian metabolism, although it has not yet been used to produce metabolites on a large scale. Here, we describe immobilisation of C. elegans as a biofilm, which can transform drugs to important human metabolites. The biofilm was cultivated on hydrophilic microtiter plates and in shake flasks containing a steel spring in contact with the glass. Fluorescence and confocal scanning laser microscopy revealed that the biofilm was composed of a dense network of hyphae, and biochemical analysis demonstrated that the matrix was predominantly polysaccharide. The medium composition was crucial for both biofilm formation and biotransformation of flurbiprofen. In shake flasks, the biofilm transformed 86% of the flurbiprofen added to hydroxylated metabolites within 24 h, which was slightly more than planktonic cultures (76%). The biofilm had a longer effective lifetime than the planktonic cells, which underwent lysis after 2×72 h cycles, and diluting the Sabouraud dextrose broth enabled the thickness of the biofilm to be controlled while retaining transformation efficiency. Thus, C. elegans biofilm has the potential to be applied as a robust biocatalyst for the production of human drug metabolites required for drug development.

Influence of culture medium on fungal biomass composition and biosorption effectiveness.

Zygomycetes such as Cunninghamella elegans seem to be promising biosorbents for pollutants removal from wastewaters because of their particular cell wall characteristics. In this article the effect of ten culture media on C. elegans biomass composition was investigated by means of Fourier transform infra red spectroscopy (FTIR). Biomasses grown on starches from potatoes and cereals were characterised by high amount of chitin and polysaccharides, the glucose gave rise to a biomass rich in acidic polysaccharides and lipids. By contrast, biomasses grown on corn steep liquor were poor in acidic polysaccharides and, when N sources and micronutrients were added, rich in proteins. The lipid content of the biomass generally increased by halving nutrients. Biosorption yields of these biomasses towards four wastewater models were assessed in terms of colour, salts and toxicity reduction. The biomasses rich in proteins and acid polysaccharides were less effective in removing reactive and direct dyes, whereas those rich in cationic polysaccharides showed a higher affinity for these dyes. Both chromatography and FTIR analyses showed that biomasses cultured in halved C and N had the highest affinity for salts. The wastewaters detoxification was quite always achieved, with values often lower that the Italian legal threshold limit.

Lipids containing polyunsaturated fatty acids synthesized by zygomycetes grown on glycerol.

Several strains of Zygomycetes cultivated on glycerol produced mycelia rich in lipids containing higher amounts of neutral lipids (NL) than glycolipids plus sphingolipids and phospholipids (P), while biosynthesis of P in Mortierella ramanniana, Mucor sp., and Cunninghamella echinulata occurred though NL accumulation process was in progress. Polyunsaturated fatty acids (PUFA) concentration gradually decreased in all lipid fractions of M. ramanniana during growth. In contrast, in C. echinulata concentration of both linoleic and γ-linolenic acids increased with time, especially in P. Taking for granted that the main function of PUFA is associated to their participation in mycelial membranes, we could suppose that biosynthesis of these fatty acids is associated to mycelial growth. However, this is accurate only for some Zygomycetes, e.g., M. ramanniana. On the contrary, PUFA biosynthesis in C. echinulata persists after growth cessation, suggesting that in this species biosynthetic ability is not a strictly growth-associated process. Phosphatidyl-inositol and phosphatidyl-choline were the major P classes in C. echinulata and M. ramanniana, respectively. In M. ramanniana, a decrease of PUFA concentration was noticed even when mycelia were incubated in low temperature (conditions that normally favor PUFA biosynthesis), indicating that PUFA biosynthesis in this fungus is associated to primary metabolism.

Cunninghamella elegans biomass optimisation for textile wastewater biosorption treatment: an analytical and ecotoxicological approach.

The effect of pre-treatments on the composition of Cunninghamella elegans biomass and on its biosorption yields in the treatment of simulated textile wastewaters was investigated. The inactivated biomass was subjected to physical treatments, such as oven drying and lyophilisation, and chemical treatments using acid or alkali. The wastewater colour, COD and toxicity variations were evaluated. The lyophilisation sped up the biosorption process, whereas the chemical pre-treatment changed the affinity of biomass for different dyes. The alkali per-treated biomass achieved the highest COD reduction in the treatment of alkali wastewaters, probably because no release of alkali-soluble biomass components occurred under the alkaline pH conditions. Accordingly, only the acid pre-treated biomass decreased the COD of the acidic effluent. The ecotoxicity test showed significant toxicity reduction after biosorption treatments, indicating that decolourisation corresponds to an actual detoxification of the treated wastewaters. Fourier transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analyses of biomasses allowed highlighting their main chemical and physical properties and the changes induced by the different pre-treatments, as well as the effect of the chemical species adsorbed from wastewaters.

Decolorization of malachite green by cytochrome c in the mitochondria of the fungus Cunninghamella elegans.

We studied the decolorization of malachite green (MG) by the fungus Cunninghamella elegans. The mitochondrial activity for MG reduction was increased with a simultaneous increase of a 9-kDa protein, called CeCyt. The presence of cytochrome c in CeCyt protein was determined by optical absorbance spectroscopy with an extinction coefficient (E(550-535)) of 19.7+/-6.3 mM(-1) cm(-1) and reduction potential of + 261 mV. When purified CeCyt was added into the mitochondria, the specific activity of CeCyt reached 440 +/- 122 micromol min(-1) mg(-1) protein. The inhibition of MG reduction by stigmatellin, but not by antimycin A, indicated a possible linkage of CeCyt activity to the Qo site of the bc1 complex. The RT-PCR results showed tight regulation of the cecyt gene expression by reactive oxygen species. We suggest that CeCyt acts as a protein reductant for MG under oxidative stress in a stationary or secondary growth stage of this fungus.

Micoparasitismo biotrofico de Fusarium oxysporum sobre Cunnighamella sp / Biotrophic mycoparasitism of Fusarium oxysporum on Cunnighamella sp

Se describe un caso de micoparasitismo biotrófico de ocurrencia natural en el suelo, entre las hifas de una cepa de Fusarium oxysporum complex y Cunninghamella sp. Las hifas de F. oxysporum se desarrollaron sobre las células vivas del hospedador, mostrando 2 tipos de efectos parasíticos: uno de enrollamiento y otro de contacto con penetración de las hifas, sin la aparente eliminación del hospedador. Esta situación poco común en la literatura, demuestra las capacidades adaptativas de esta especie al micoparasitismo en grupos filogenéticamente distantes.

This paper describes a case of mycoparasitism naturally occurring, where Fusarium oxysporum parasitizes hyphae of Cunninghamella sp, to show mycoparasitism between the two fungi. This is a case of biotrophic mycoparasitism by contact. The hyphae of F. oxysporum developed closely along the living cells of the host showing mycoparasitic effect, some for a loop, and other contact with penetration of the hyphae. This situation is rare in the literature, demonstrates the adaptive capacities of this species to mycoparasitism in phylogenetically distant groups.

Responses of fungi to tropane alkaloids produced by a medicinal plant Hyoscyamus muticus (Egyptian henbane).

Antifungal activity of hyoscyamine (Hcy) and scopolamine (Sco) were determined by TLC-bioautography against fungi associated with H. muticus grown in Egypt, and those isolated from other plants grown in Japan. All 40 fungal strains were tolerant to Sco and sensitive to Hcy, exhibiting a growth inhibition zone around the Hcy spot on the bioautography plate. The strains were grouped into three types based on the appearance of the inhibition zone: (i) 17 strains exhibiting a clear inhibition zone, which remained clear at 8 d after incubation (type I); (ii) 22 strains exhibiting the inhibition zone with a brown circle surrounding the zone and regrowth within the inhibition zone (type II); (iii) 1 strain exhibiting the inhibition zone with no brown circle and regrowth within the inhibition zone (type III). In the type II and III strains, Hcy disappeared, and other alkaloids were found in the inhibition zones in its place. Hcy feeding experiments using Penicillium purpurogenum (type II) and Cunninghamella elegans (type III) revealed that these fungi may convert Hcy to a new alkaloid compound.

[Process fundamentals and field demonstration of wheat straw enhanced biodegradation of petroleum].

A new bioaugmentation method utilizing wheat straw to enhance salt leaching and the subsequent petroleum biodegradation by consortia of bacteria and fungi was proposed. The present study aimed at the effects of wheat straw on the growth and the degradation behavior of E. cloacae and Cun. echinulata, the two species of the consortia. In the laboratory experiments, it was shown that the addition of 5% (mass fraction) straw led to an increase of biomass by 25- and 3-fold to the bacteria and fungi, respectively. The biodegradation ratio of total petroleum hydrocarbon (TPH) was elevated from 29.2% to 48.0% after 468 h treatment. The biodegradation ratio of alkane and aromatic hydrocarbons in petroleum were increased from 31.5% and 39.1%, to 55.7% and 55.9%, respectively. The field demonstration was carried in an area of 6400 m2, in which the bacteria and fungi were inoculated after salt leaching in the presence of wheat straw. The addition of wheat straw in the contaminated soil led to an increase by 158- and 9-fold to the bacteria and fungi, as compared to their counterpart in the controlland without wheat straw, at 25 days after the inoculation. The content of TPH was down to below 0.3% while the maximum biodegradation ratio of TPH reached 75% after 45 days treatment. These results demonstrated the effectiveness and high potential of the wheat straw enhanced bioaugmentation of petroleum-salt contaminated soil.