Growth performance of Ganoderma lucidum using billet method in Garhwal Himalaya,India

Medicinal mushrooms have been used in various treatments from a very long time, among which, Ganoderma lucidum is one of the most important medicinal mushroom. It is cultivated worldwide to meet its ever-increasing demand in the market. It is generally cultivated by bed log (Sawdust) and wood log (billet) method. This study was an attempt to observe the growth performance of G. lucidum on poplar billets (Populus deltoides) in the Sherpur Village (Dehradun) and Manjgaun village (Tehri Garhwal) of Garhwal Himalaya, India. The farmers’ field with empty house/ rooms having proper growing conditions especially humidity and light were used for the cultivation of G. lucidum. The G. lucidum spawn was inoculated in poplar wood billets and these billets were installed in well prepared soil. The results demonstrated that cropping cycle of G. lucidum was shorter (132–136 days) in Sherpur Village (Dehradun) as compared to Manjgaun village (141–145 days) in Tehri Garhwal. Further the results also revealed that yield was decreased in the subsequent flushes. In Village Sherpur, the fruiting bodies of G. lucidum were harvested between 64-66 days, 100-101  days and 135-136  days during first, second and third flush after the installation of billets, respectively. However; in village Manjgaun, the fruiting bodies of G. lucidum were harvested between 69 and 71 days, 107-108  days and 144-145 days in first, second and third after the installation of billets respectively. Warmer temperature in Village Sherpur resulted in the early emergence and development of the fruiting bodies as compared to village Manjgaun where pinhead and fruiting body development was delayed due to the lower temperature during cropping cycle.     

Complete Mitochondrial Genome of the Medicinal Mushroom Ganoderma lucidum

Ganoderma lucidum is one of the well-known medicinal basidiomycetes worldwide. The mitochondrion, referred to as the second genome, is an organelle found in most eukaryotic cells and participates in critical cellular functions. Elucidating the structure and function of this genome is important to understand completely the genetic contents of G. lucidum. In this study, we assembled the mitochondrial genome of G. lucidum and analyzed the differential expressions of its encoded genes across three developmental stages. The mitochondrial genome is a typical circular DNA molecule of 60,630 bp with a GC content of 26.67%. Genome annotation identified genes that encode 15 conserved proteins, 27 tRNAs, small and large rRNAs, four homing endonucleases, and two hypothetical proteins. Except for genes encoding trnW and two hypothetical proteins, all genes were located on the positive strand. For the repeat structure analysis, eight forward, two inverted, and three tandem repeats were detected. A pair of fragments with a total length around 5.5 kb was found in both the nuclear and mitochondrial genomes, which suggests the possible transfer of DNA sequences between two genomes. RNA-Seq data for samples derived from three stages, namely, mycelia, primordia, and fruiting bodies, were mapped to the mitochondrial genome and qualified. The protein-coding genes were expressed higher in mycelia or primordial stages compared with those in the fruiting bodies. The rRNA abundances were significantly higher in all three stages. Two regions were transcribed but did not contain any identified protein or tRNA genes. Furthermore, three RNA-editing sites were detected. Genome synteny analysis showed that significant genome rearrangements occurred in the mitochondrial genomes. This study provides valuable information on the gene contents of the mitochondrial genome and their differential expressions at various developmental stages of G. lucidum. The results contribute to the understanding of the functions and evolution of fungal mitochondrial DNA.     

Preservation affects the vegetative growth and fruiting body production of <Emphasis Type="Italic">Cordyceps militaris</Emphasis>

Cordyceps militaris is a model species of Cordyceps fungi, and has been traditionally used as an edible and medicinal fungus due to its richness of bioactive and pharmacological metabolites. The fruiting bodies of this fungus are widely used as healthy food and nutrition supply. In industrial production, fruiting bodies are cultivated on artificial media, but their yield and quality are usually affected by the quality of fungal strains. In this study, the effect of colony growth rate of the fungal strains, fungal age and repeated subculturing on the fungal biomass accumulation was investigated. The results indicated that the fungal biomass was positively correlated with the colony growth rate and not affected by fungal age and the repeated subculturing. The preservation conditions for stock cultures, including choice of cultures, lyophilization, temperature and protective agents were optimized based on the mycelial formation and conidia production in artificial inoculum. The development of fruiting bodies from the fungal strains stored under the optimized preservation conditions were further analyzed to determine the ideal time period of preservation. Results indicated that storing the fungus at 4 °C could maintain the fungal vitality and fruiting body producing capacity for at least 12 months. This study established practical criteria of fungal inoculum for artificial cultivation of fruiting body and provided a simple and efficient preservation method for C. militaris. The results may shed light on preservation for other Cordyceps species and other edible fungi.     

Ganoderma lucidum: A potential pleiotropic approach of ganoderic acids in health reinforcement and factors influencing their production

Ganoderma lucidum (G. lucidum) main attractive pharmacological characteristics are antitumor and immunomodulatory activities which are chiefly associated with its two principal bioactive compounds, those are polysaccharides and triterpenoids. Ganoderic acids (GAs) are one of the most discovered triterpenoids of G. lucidum among various triterpenoids. The prominent medicinal mushroom G. lucidum possesses GAs as essential bioactive constituents that are highly oxygenated lanostane-type triterpenoids. GAs exhibit diverse potential action against numerous diseases such as anticancer, antioxidant, anti-inflammatory, anti-HIV, cardioprotective, antiallergic, antihepatotoxic, neuroprotective and antinociceptive properties. GAs act through different mechanisms that include cytotoxic, apoptosis, inducing cell cycle arrest, inhibition of topoisomerases, antiproliferation, anti-invasion, inhibition of NF-kB AP1/uPA, farnesyl protein transferase and JAK-STAT3 pathway. The miraculous effects of GAs fascinate the researchers for their production. Various environmental factors such as biochemical signals, nutritional and physical that influence the biosynthesis of GA. However, the scarcities of pure compounds or accurately characterized extracts are the main problem of clinical studies. Substantial steps are required for characterized extracts of active compounds. This review contributes a thorough insight into the mode of actions of GAs and their possible reinforcements to overcome various diseases.     

Biochemical constituents of a wild strain of Schizophyllum commune isolated from Achanakmar-Amarkantak Biosphere Reserve (ABR), India

A wild strain of Schizophyllum commune (MTCC 9670) isolated from Achanakmar-Amarkantak Biosphere Reserve of Central India was evaluated for the production of bioactive compounds. The chemical constituents of wild and in vitro grown cultures were compared. Under optimized conditions, different organic and aqueous extracts from mycelia and fruiting bodies were used to extract chemical components from the cultures grown in vitro. The gas chromatography combined wih mass spectrometry analysis of extracts identified two phenolic compounds, namely Phenyl benzoate (C₁₃H₁₀O₂) and 4-(phenyl methoxy) phenol (C₁₃H₁₂O₂) in the ethanolic extract of in vitro grown fruiting bodies and one antibacterial compound Pyrrolo (1, 2-a) piperazine-3, 6-dione (C₇H₁₀O₂N₂) in the methanolic extract of mycelia. High-performance liquid chromatography analysis revealed that the gallic acid and l-ascorbic acid were identifiable antioxidant components in the extracts possessing high free radical scavenging activity. The findings suggest that the wild strain of S. commune may serve as the source of novel bioactive compounds with effective antimicrobial and antioxidant activities.     

Ganoderol B: A potent α-glucosidase inhibitor isolated from the fruiting body of Ganoderma lucidum

α-Glucosidase inhibitor has considerable potential as a diabetes mellitus type 2 drug because it prevents the digestion of carbohydrates. The search for the constituents reducing α-glucosidase activity led to the finding of active compounds in the fruiting body of Ganoderma lucidum. The CHCl₃ extract of the fruiting body of G. lucidum was found to show inhibitory activity on α-glucosidase in vitro. The neutral fraction, with an IC₅₀ of 88.7 μg/ml, had stronger inhibition than a positive control, acarbose, with an IC₅₀ of 336.7 μg/ml (521.5 μM). The neutral fraction was subjected to silica gel column chromatography and repeated p-HPLC to provide an active compound, (3β,24E)-lanosta-7,9(11),24-trien-3,26-diol (ganoderol B). It was found to have high α-glucosidase inhibition, with an IC₅₀ of 48.5 μg/ml (119.8 μM).     

The identification of active compounds in Ganoderma lucidum var. antler extract inhibiting dengue virus serine protease and its computational studies

Abstract

The number of global dengue incidences is alarmingly high in recent years. The global distribution of four dengue serotypes has also added economic burden in the dengue-endemic countries. To discover the potent dengue virus inhibitors in the antler form of Ganoderma lucidum (Lingzhi or Reishi), the water extraction of normal G. lucidum and its antler form were conducted and the chemical compounds were identified by LC-MS. Six distinct chemical compounds identified in high abundance were hesperetin, thymidine, lucidenic acid, 11-aminoundecanoic acid, 5-carboxyvanillic acid and ganocin B. The water extracts of G. lucidum in its antler form inhibited the DENV2 NS2B-NS3 protease activity at 84.6?±?0.7%, higher than the normal G. lucidum. Then, molecular docking was performed on the homology model built from an in-house sequence. Docking simulation results showed that hesperetin and ganocin B were the best leads to bind at the catalytic triad of DENV2 NS2B-NS3pro via hydrogen bonding, van der Waals and pi-pi interactions. Extensive overlapping of HOMO-LUMO orbitals at the ringed regions of hesperetin helped to facilitate the entry of ligand to the catalytic triad cleft. LC-MS, molecular docking and density functional theory analyses confirmed that hesperetin was the strongest inhibitor against NS2B-NS3 protease.

Communicated by Ramaswamy H. Sarma     

Purification and characterization of lectin from fruiting body of Ganoderma lucidum: Lectin from Ganoderma lucidum

A novel 114 kDa hexameric lectin was purified from the fruiting bodies of the mushroom Ganoderma lucidum. Biochemical characterization revealed it to be a glycoprotein having 9.3% neutral sugar and it showed hemagglutinating activity on pronase treated human erythrocytes. The lectin was stable in the pH range of 5–9 and temperature up to 50 °C. The hemagglutinating activity was inhibited by glycoproteins that possessed N-as well as O-linked glycans. Chemical modification of the G. lucidum lectin revealed contribution of tryptophan and lysine to binding activity. The thermodynamics of binding of bi- and triantennary N-glycans to G. lucidum lectin was studied by spectrofluorimetry. The lectin showed very high affinity for asialo N-linked triantenary glycan and a preference for asialo glycans over sialylated glycans. The binding was accompanied with a large negative change in enthalpy as well as entropy, indicating primarily involvement of polar hydrogen, van der Waals and hydrophobic interactions in the binding.     

Formation of atypical fruiting structures inGanoderma lucidum isolates on a nutrient agar medium

Effects of light and ventilation on the formation of atypical fruiting structures (AFSs) and fruit body primordia (FBPs) ofGanoderma lucidum on nutrient agar media were investigated. Although the mycelial growth was inhibited by illumination and ventilation, brown AFSs appeared on the white mycelial colony, and basidia and basidiospores were produced on the AFSs. On the other hand, FBPs were induced by illumination alone, regardless of ventilation. However, the primordia could not develop to mature fruit bodies. In the dark, only vegetative growth of the fungus progressed. Twenty-three isolates ofG. lucidum collected from four countries were tested for the formation of AFSs and FBPs under light and ventilation. Thirteen isolates formed AFSs, and another five isolates produced FBPs. Of the remaining five isolates, one formed callus-like structures without elaborating basidiospores, and the other four did not induce AFSs or FBPs. Microscopical observation showed that the basidia were formed directly from generative hyphae on the surface of AFSs. Basidiospores formed on the basidia were brown and ellipsoid with an eccentric hilar appendix on the rounded spore base. They had a double wall and mostly contained one or two large vacuoles. The surface of basidiospores was smooth or wrinkled and had shallow holes. The spore size was (4.5–)6.4–9.6(–10.3) × (2.6–)3.2–5.1(–6.4), 7.3 × 4.2 µm on average.     

Characterization of vitamin B12 compounds in the fruiting bodies of shiitake mushroom (Lentinula edodes) and bed logs after fruiting of the mushroom

This study determined the vitamin B₁₂ content in commercially available dried fruiting bodies of shiitake mushroom, Lentinula edodes. The vitamin B₁₂ contents in dried donko-type fruiting bodies with closed caps (5.61 ± 3.90 μg/100 g dry weight), did not significantly differ from those of dried koushin-type fruiting bodies with open caps (4.23 ± 2.42 μg/100 g dry weight). The bed logs after fruiting of the mushroom also contained the vitamin B₁₂ levels similar to that in the dried shiitake fruiting bodies. To determine whether the dried shiitake fruiting bodies and their bed logs contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified corrinoid compounds using an immunoaffinity column and identified vitamin B₁₂ using vitamin B₁₂-dependent Escherichia coli 215 bioautograms and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) chromatograms. Dried shiitake fruiting bodies rarely contained an unnatural corrinoid vitamin B₁₂[c-lactone] that is inactive in humans. Given that shiitake mushroom lacks the ability to synthesize vitamin B₁₂ de novo, the vitamin B₁₂ found in dried shiitake fruiting bodies must have been derived from the bed logs.     

Effect of light on the formation of atypical fruiting structures inGanoderma lucidum

Ganoderma lucidum develops atypical fruiting structures (AFSs) with non-basidiocarpous basidiospores during the incubation under light on nutrient agar media. To examine the light quality effective in inducing AFSs, 17 isolates ofG. lucidum were incubated on agar media under light from different colored fluorescent lamps. Of the 17 isolates, 13 isolates produced AFSs and basidiospores under fluorescent lamps. Nine isolates formed AFSs in a broad light region from P-B (pure blue) to P-R (pure red) lamps. The remaining 4 isolates produced AFSs under different colored fluorescent lamps. No isolates formed AFSs in the dark or under BLB (black light blue) illumination. The mycelial growth was inhibited by light illumination, especially BLB light. Although the AFSs were induced at a very low light intensity such as 0.5µmol m^–2s^–1, the optimum light intensity for the AFS formation varied depending on the kind of fluorescent lamp and the isolate. The AFS formation inG. lucidum isolates was also tested under monochromatic light produced by the combination of interference filters and colored glass filters.G. lucidum isolates were separable into various types in the responses of AFS formation to monochromatic light, indicating thatG. lucidum is heterogeneous in its photo-response with regard to AFS formation.     

Photosynthetic and Respiratory Activity of Fruiting Forms within the Cotton Canopy

The supply of photosynthates by leaves for reproductive development in cotton (Gossypium hirsutum L.) has been extensively studied. However, the contribution of assimilates derived from the fruiting forms themselves is inconclusive. Field experiments were conducted to document the photosynthetic and respiratory activity of cotton leaves, bracts, and capsule walls from anthesis to fruit maturity. Bracts achieved peak photosynthetic rates of 2.1 micromoles per square meter per second compared with 16.5 micromoles per square meter per second for the subtending leaf. However, unlike the subtending leaf, the bracts did not show a dramatic decline in photosynthesis with increased age, nor was their photosynthesis as sensitive as leaves to low light and water-deficit stress. The capsule wall was only a minor site of ¹⁴CO₂ fixation from the ambient atmosphere. Dark respiration by the developing fruit averaged −18.7 micromoles per square meter per second for 6 days after anthesis and declined to −2.7 micromoles per square meter per second after 40 days. Respiratory loss of CO₂ was maximal at −158 micromoles CO₂ per fruit per hour at 20 days anthesis. Diurnal patterns of dark respiration for the fruit were age dependent and closely correlated with stomatal conductance of the capsule wall. Stomata on the capsule wall of young fruit were functional, but lost this capacity with increasing age. Labeled ¹⁴CO₂ injected into the fruit interior was rapidly assimilated by the capsule wall in the light but not in the dark, while fiber and seed together fixed significant amounts of ¹⁴CO₂ in both the light and dark. These data suggest that cotton fruiting forms, although sites of significant respiratory CO₂ loss, do serve a vital role in the recycling of internal CO₂ and therein, function as important sources of assimilate for reproductive development.     

Ganoderma lucidum inspired silver nanoparticles and its biomedical applications with special reference to drug resistant Escherichia coli isolates from CAUTI

In the search for alternative therapy for infections and other ailments, metallic nanoparticles, mainly silver nanoparticles (AgNPs) synthesized through bioengineered sources are extensively explored. Fungal bioactive compounds and their nanoparticles were reported with the potential biomedical application. A medicinal mushroom Ganoderma lucidum was reported as a repository of rich medicinal properties. In the current study, silver nanoparticles were synthesized using the extracts of G. lucidum and its antimicrobial activity was tested against drug-resistant Escherichia coli isolated from the catheter used for urinary tract infection (CAUTI). The GC–MS study of G. lucidum extracts showed the presence of ethyl acetoacetate ethylene acetal with the highest area percentage of 72.2% and retention time (RT 5873). Pyridine-3-ol is the second primary compound with a peak height of 6.44% and a retention time of 2.143. The third compound is l,4-Dioxane-2,3-diol, with an area of 8.09% and RT 5450. Butylated Hydroxy Toluene [BHT] is the fourth major compound with an area of 3.32%, and 9-Cedranone constitutes the fifth position in occupying the area percentage [1.88] and height 1.56%. Pyrrole is the sixth primary compound registering an area size of 0.96% and height 2.06%. The AgNPs synthesized using G. lucidum extract were in size range 23 and 58 nm as per SEM analysis and within the range wavelength 0.556–0.796 nm as per UV–Vis spectral study. FTIR Spectroscopy and X-ray diffraction analysis (XRD) were made to characterize the formed nanoparticles. The AgNPs synthesized effectively inhibited the growth of E. coli isolated from catheter-associated urinary tract infection and showed resistance to many drugs. The antioxidant potential of the synthesized nanoparticles assessed using DPPH radical scavenging activity, EC50 (µg/ml), and ARP data showed that the prepared nanoparticles were more potent in free radical scavenging activity than the standard quercetin. The cytotoxicity effect of Ag-NPs on breast cancer cell line- MDA-MB-231 confirmed its anticancer potential. The half-maximal inhibitory concentration (IC₅₀) of Ag-NPs to inhibit 50% of the tumor was 9.2 g/mL. The synthesized GL-AgNPs was exhibited a multifocal biomedical potential.     

Biochemical characterization of cultivated Cordyceps bassiana mycelia and fruiting bodies by 1H nuclear magnetic resonance spectroscopy

In this study, nuclear magnetic resonance techniques coupled with multivariate data analysis were used for the metabolic profiling of mycelia and fruiting bodies of the entomopathogenic fungi, Cordyceps bassiana according to developmental stages. A direct extraction method using two deuterated solvents of D₂O and CDCl₃ was used to investigate the relative levels of identified metabolites in each extraction condition in the mycelium and fruiting body formation stages. There was a clear separation among mycelia and fruiting bodies with various developmental stages in partial least-squares discriminant analysis (PLS-DA) derived score plots. During the transition from mycelia to fruiting bodies, the major metabolic change observed was the conversion of glucose to mannitol, and beauvericin to phenylalanine and 1-hydroxyisovaleric acid. In the developmental stages of fruiting bodies studied, there was a clear separation between stage 3 and the other stages in PLS-DA derived score plots. Nineteen compounds including 13 amino acids, 2 nucleosides, 3 organic acids, and glucose showed the highest levels in stage 3 fruiting bodies. The flavonoid content in the fruiting bodies showed similar levels during stages 1, 2, and 3, whereas the level at stage 4 was significantly decreased compared to the other stages. Results suggest that the fruiting body of C. bassiana is richer in natural resources at stage 3 compared to the other fruiting body stages due to its high abundance of compounds including total flavonoids. The metabolome information acquired in this study can be useful criteria for the quality control of commercial use of C. bassiana.     

Lactarius deliciosus and Pinus radiata in New Zealand: towards the development of innovative gourmet mushroom orchards

The cultivation of Lactarius deliciosus (saffron milk cap) in New Zealand began in 2002 when fruiting bodies were produced in an Otago plantation of Pinus radiata seedlings artificially mycorrhized by L. deliciosus. In 2007, 42 P. radiata seedlings mycorrhized by L. deliciosus under controlled conditions were planted in a grass field at Plant and Food Research (Lincoln, Canterbury). The effects of pine bark mulch application and initial degree of mycorrhization of seedlings were examined to determine their influence on tree growth, development of mycorrhizae (i.e. their multiplication on the root system and their degree of branching) and fruiting body production. Mulch application increased tree growth significantly over 4 years. High initial mycorrhization slightly stimulated tree growth over 2 years. The initial degree of mycorrhization was positively, but not strongly, related to the persistence and development of L. deliciosus mycorrhizae and rhizomorphs based on root sample analyses 2 years after planting. However, mulching strongly reduced the proportion of highly branched L. deliciosus mycorrhizae compared with poorly ramified ones. A positive correlation was observed between the fruiting of L. deliciosus and the development of mycorrhizae. Mulching delayed the onset of fruiting body production. In 2010, fruiting bodies were produced only from non-mulched trees with eight of these (38 %) producing a total of 12 fruiting bodies. In 2011, 19 non-mulched trees (90 %) and 9 mulched trees (45 %) produced 143 and 47 fruiting bodies, respectively, totalling 190 fruiting bodies. By 2012, 19 non-mulched trees (90 %) and 13 mulched trees (65 %) produced 333 and 236 fruiting bodies, respectively, totalling 569 fruiting bodies (c. 30 kg). This study presents new information on factors influencing the onset of fruiting and the development of yields in a plantation of P. radiata mycorrhized by L. deliciosus. Projected yields as high as c. 300 kg/ha from the third year of production reiterate the feasibility of farming saffron milk cap in P. radiata plantations in New Zealand. Continued monitoring of this site and development of similar trials will provide important knowledge for the optimisation of yields in commercial saffron milk cap orchards.     

Tea waste as a supplement for the cultivation of <Emphasis Type="Italic">Ganoderma</Emphasis> <Emphasis Type="Italic">lucidum</Emphasis>

Tea waste (TW) was investigated as a new supplement for substrate mixtures in Ganoderma lucidum cultivation. The effects of sawdust (S) based substrates supplemented with TW at the various levels (75S:25TW, 80S:20TW, 85S:15TW, and 90S:10TW) and Ganoderma lucidum strains on yield, biological efficiency (BE) and the chemical composition of fruiting bodies were determined in solid-state fermentation. Significant differences were found among substrates regarding yield and BE, while yield and BE of the strains were not different. The substrate formulations producing highest yield and BE were 80S:20TW (87.98 g/kg substrate and 34.90%) and 75S:25TW (82.30 g/kg substrate and 31%). Yield and BE of substrates containing TW were generally higher than that of the control (80sawdust:18wheat bran:1sucrose:1 CaCO₃). Nitrogen, potassium, iron, and manganese contents and C:N ratios of substrates were strongly correlated with yield. BE showed positive and significant correlations with potassium, iron and manganese. Moisture content, potassium, magnesium, calcium, iron, and zinc contents of the fruiting bodies were affected by both strain and substrate. It was concluded that TW can be used as a supplement for substrate preparation in G. lucidum cultivation.     

Distribution of free and esterified ergosterols in the medicinal fungus <Emphasis Type="Italic">Ganoderma lucidum</Emphasis>

The fruiting bodies, spores, and lipid from the spores of Ganoderma lucidum have been widely used for medicinal purpose in China. Ergosterol content may be a suitable marker for evaluating the quality of ganoderma spore and ganoderma spore lipid (GSL) products. A gradient reversed-phase high-performance liquid chromatography method was developed for the simultaneous determination of free and esterified ergosterols in G. lucidum. The contents of free and esterified ergosterols in the different parts (the stipe, pileus, tubes, and spores) of G. lucidum and GSL were determined. The results showed that total ergosterol levels in the stipe, pileus, tubes, and spores of G. lucidum were between 0.8 and 1.6 mg/g. The relative abundances of free to esterified ergosterol were different in the different parts of G. lucidum. The spores and the tubes, the hymenophore tissue that contains the spore-producing cells, have a considerably higher percentage of ergosteryl esters (41.9 and 39.7% of total ergosterol) in comparison with the pileus and stipe tissues (3.6 and 6.2%).     

Use of lignocellulosic wastes of pecan (Carya illinoinensis) in the cultivation of Ganoderma lucidum

Background

The wastes of pecan nut (Carya illinoinensis (Wangenh.) K. Koch) production are increasing worldwide and have high concentrations of tannins and phenols.

The role of Gracilaria lemaneiformis in eliminating the dissolved inorganic carbon released from calcification and respiration process of Chlamys farreri

Respiration and calcification rate were estimated to quantify the effect of Zhikong scallop Chlamys farreri on marine CO₂ system in Sanggou Bay, China. The C. farreri population in Sanggou Bay sequestered 78.06?±?5.76 g C m^?2 y^?1 for shell formation, while the CO₂ fluxes due to calcification and respiration were 53.95?±?3.98 and 71.69?±?6.51 g C m^?2 y^?1, respectively. In order to eliminate the additional CO₂ released from calcification and respiration process of C. farreri, Gracilaria lemaneiformis were introduced into the integrated system and its role was validated by in situ mesocosm methods. Eight mesocosms (1,000 L) were deployed over 42-h period and consisted of four treatments: seaweed-only, scallop-only (SP), seaweed integrated with scallop (SS), and control (C). The aqueous CO₂ concentration and partial pressure of CO₂ in SP treatments were significantly higher than the other three treatments (p?<?0.01), while there were no difference between SS treatments and C treatments (p?>?0.05). Furthermore, compared with the SP treatments, the presence of the G. lemaneiformis can keep the seawater pH stable. These findings suggest that seaweed and shellfish integrated aquaculture practice cannot only reduce dissolved inorganic carbon but also can alleviate ocean acidification.