Biological characterization and complete genome analysis of the newly isolated Serratia liquefaciens phage vB_SlqS_ZDD2.

A novel lytic phage named vB_SlqS_ZDD2 was isolated from hospital sewage using the double-layer agar method with Serratia liquefaciens ATCC 27592 as the host. BLASTn analysis showed that the genome sequence of phage vB_SlqS_ZDD2 did not resemble any other phages in the NCBI database. Phenotype and phylogeny analysis indicated that this phage might be a new member of the class Caudoviricetes. Phage vB_SlqS_ZDD2 has a dsDNA genome of 49,178 bp with 55% GC content and has 73 open reading frames. This phage exhibited strong lytic activity and a wide range of pH (3-12) and temperature tolerance (below 70℃).

Genomic analysis and biological properties of the novel Serratia liquefaciens phage vB_SlqM_MQ-4.

A novel lytic Serratia liquefaciens phage, named vB_SlqM_MQ-4, was isolated from sewage. BLASTn analysis showed that the genome sequence of phage vB_SlqM_MQ-4 shared only 15% query coverage with that of Escherichia phage vB_EcoM-ep3, with 80.52% identity. Genomic analysis demonstrated that phage vB_SlqM_MQ-4 has a 43,534-bp dsDNA genome with 56% GC content and might be a member of a new genus in the order Caudoviricetes. Moreover, vB_SlqM_MQ-4 exhibited strong lytic performance with a short latent period (10 min) and a high burst size (267 PFU per cell) as well as a wide range of thermal (below 70 ℃) and pH tolerance (pH 4-12).

Phenolic extract of Eugenia uniflora L. and furanone reduce biofilm formation by Serratia liquefaciens and increase its susceptibility to antimicrobials.

Serratia liquefaciens is a spoilage microorganism of relevance in the dairy industry because it is psychrotrophic, able to form biofilm, and produces thermoresistant proteases and lipases. Phenolic compounds and furanones have been studied as inhibitors of biofilm formation. In this study, the potential of the pulp phenolic extract of Eugenia uniflora L. orange fruits, also called pitanga, and furanone C30 on the inhibition of biofilm formation by S. liquefaciens L53 and the susceptibility to different antimicrobials were evaluated. The pulp phenolic extract of pitanga had a high total phenolic content, being mainly composed of glycosylated quercetins and ellagitannins. Sub-inhibitory concentrations of this extract and furanone reduced biofilm formation by S. liquefaciens on polystyrene and the amount of polysaccharides, proteins and extracellular DNA in the biofilms. These biofilms were also more susceptible to kanamycin. The combinations of furanone with phenolic extract of pitanga or kanamycin showed a synergistic effect with total growth inhibition of S. liquefaciens.

First case of Serratia liquefaciens isolated from urinary tract infection in sows and associated clinicopathological and pathological findings.

An incident of sudden deaths in the breeding stock was reported from a farrow-to-finish commercial pig farm in Greece. The 8·4% of sows during lactation and gestation period presented anorexia, fever, haematuria, return-to-oestrus and sudden deaths (mortality rate: 2·3%). Blood and urine samples were collected from four diseased sows. Furthermore, swabs from urine bladders were collected from two dead sows and four culled sows at the slaughterhouse. Blood testing demonstrated mild leucocytosis and absence of azotaemia. Urinalysis revealed haematuria, proteinuria, bilirubinuria and active urine sediment with bacilli, epithelial cells and leucocytes, crystals and granular casts. Histopathological evaluation of the bladder demonstrated chronic active polypoid cystitis. The bacterial culture revealed the presence of Serratia liquefaciens. The antibiotic susceptibility testing showed high resistance to the most common antibiotics, with the highest sensitivity of the isolate towards quinolones. After the administration of a single dose of 7·5 mg kg-1 body weight enrofloxacin intramuscularly, the mortality rate decreased to less than 0·5% along with a remarkable reduction in the severity of clinical signs. Based on our findings, S. liquefaciens induced severe clinical signs and deaths in sows, mainly due to urinary infection. Inadequate water sanitation might have been responsible for increased exposure to S. liquefaciens. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the isolation of Serratia liquefaciens from the urinary tract of pigs associated with clinical signs and increased mortality was described for the first time. Serratia liquefaciens is an important cause of hospital-acquired human infections. The isolate in this study was resistant to the most common antibiotics. Therefore, the use of quinolones which are drugs of last resort for treatment of infections was the only therapeutic option. The presence of the resistant bacterium in the urinary tract raises concerns for its zoonotic potential.

Effect of glucose, pH and lactic acid on Carnobacterium maltaromaticum, Brochothrix thermosphacta and Serratia liquefaciens within a commercial heat-shrunk vacuum-package film.

Carnobacterium maltaromaticum, Brochothrix thermosphacta and Serratia liquefaciens are common spoilage organisms found within the microbiome of refrigerated vacuum-packaged (VP) beef. Extending and predicting VP beef shelf-life requires knowledge about how spoilage bacteria growth is influenced by environmental extrinsic and intrinsic factors. Multifactorial effects of pH, lactic acid (LA) and glucose on growth kinetics were quantified for C. maltaromaticum, B. thermosphacta and S. liquefaciens within a heat shrink-wrapped VP commercial film containing a simulated beef medium. LA, pH, and undissociated lactic acid (UDLA) significantly affected bacterial growth rate (p < 0.001), whereas 5.55 mM glucose produced a marginal effect. At 1.12 mM UDLA, growth rate and maximum population density decreased 20.9 and 3.5%, 56 and 7%, and 11 and 2% for C. maltaromaticum, B. thermosphacta, and S. liquefaciens, respectively.

Heavy metal-immobilizing bacteria increase the biomass and reduce the Cd and Pb uptake by pakchoi (Brassica chinensis L.) in heavy metal-contaminated soil.

Microbial immobilization is a novel and environmentally friendly technology that uses microbes to reduce metal availability in soil and accumulation of heavy metals in plants. We used urea agar plates to isolate urease-producing bacteria from the rhizosphere soil of pakchoi in Cd- and Pb-contaminated farmland and investigated their effects on Cd and Pb accumulation in pakchoi and the underlying mechanisms. The results showed that two urease-producing bacteria, Bacillus megaterium N3 and Serratia liquefaciens H12, were identified by screening. They had higher ability to produce urease (57.5 ms cm-1 min-1 OD600-1 and 76.4 ms cm-1 min-1 OD600-1, respectively). The two strains allowed for the immobilization of Cd and Pb by extracellular adsorption, bioprecipitation, and increasing the pH (from 6.94 to 7.05-7.09), NH4+ content (69.1%-127%), and NH4+/NO3- ratio (from 1.37 to 1.67-2.11), thereby reducing the DTPA-extractable Cd (35.3%-58.8%) and Pb (37.8%-62.2%) contents in the pakchoi rhizosphere soils and the Cd (76.5%-79.7%) and Pb (76.3%-83.5%) contents in the leaves (edible tissue) of pakchoi. The strains were highly resistant to heavy metal toxicity; produced IAA, siderophores and abscisic acid; and increased the NH4+/NO3- ratio, which might be related to the two strains protectiing pakchoi against the toxic effect of Cd and Pb and increasing pakchoi biomass. Thus, the results were supposed to strain resources and a theoretical basis for the remediation of Cd- and Pb-contaminated farmlands for the safe production of vegetables.

Combined biochar and metal-immobilizing bacteria reduces edible tissue metal uptake in vegetables by increasing amorphous Fe oxides and abundance of Fe- and Mn-oxidising Leptothrix species.

In this study, a highly effective combined biochar and metal-immobilizing bacteria (Bacillus megaterium H3 and Serratia liquefaciens CL-1) (BHC) was characterized for its effects on solution Pb and Cd immobilization and edible tissue biomass and Pb and Cd accumulation in Chinese cabbages and radishes and the mechanisms involved in metal-polluted soils. In the metal-containing solution treated with BHC, the Pb and Cd concentrations decreased, while the pH and cell numbers of strains H3 and CL-1 increased over time. BHC significantly increased the edible tissue dry weight by 17-34% and reduced the edible tissue Pb (0.32-0.46 mg kg-1) and Cd (0.16 mg kg-1) contents of the vegetables by 24-45%. In the vegetable rhizosphere soils, BHC significantly decreased the acid-soluble Pb (1.81-2.21 mg kg-1) and Cd (0.40-0.48 mg kg-1) contents by 26-47% and increased the reducible Pb (18.2-18.8 mg kg-1) and Cd (0.38-0.39 mg kg-1) contents by 10-111%; while BHC also significantly increased the pH, urease activity by 115-169%, amorphous Fe oxides content by 12-19%, and relative abundance of gene copy numbers of Fe- and Mn-oxidising Leptothrix species by 28-73% compared with the controls. These results suggested that BHC decreased edible tissue metal uptake of the vegetables by increasing pH, urease activity, amorphous Fe oxides, and Leptothrix species abundance in polluted soil. These results may provide an effective and eco-friendly way for metal remediation and reducing metal uptake in vegetables by using combined biochar and metal-immobilizing bacteria in polluted soils.

Metal-immobilizing and urease-producing bacteria increase the biomass and reduce metal accumulation in potato tubers under field conditions.

In this study, the effect of two metal-immobilizing bacterial strains, Serratia liquefaciens CL-1 and Bacillus thuringiensis X30, on the availability of Cd and Pb and the metal accumulation in potato tubers, as well as the underlying mechanisms in metal-contaminated soils were characterized. Moreover, the impacts of the strains on metal immobilization, pH, and NH4+ concentration in metal-contaminated soil solutions were evaluated. Strains CL-1 and X30 increased tuber dry weight by 46% and 40%, reduced tuber Cd and Pb contents by 68-83% and 42-47%, and decreased the Cd and Pb translocation factors by 61-70% and 30-34%, respectively, compared to the controls. Strains CL-1 and X30 decreased the available Cd and Pb contents by 52-67% and 30-44% and increased the NH4+ content by 55% and 31%, pH, urease activity by 70% and 41%, and relative abundance of ureC gene copies by 37% and 20% in the rhizosphere soils, respectively, compared with the controls. Reduced Cd and Pb concentrations and increased pH and NH4+ concentration were found in the bacteria-inoculated soil solution compared to the controls. These results suggested that the strains reduced tuber metal uptake through decreasing the metal availability and increasing the pH, ureC gene relative abundance and urease activity as well as decreasing the metal translocation from the leaves to tubers. These results may provide an effective metal-immobilizing bacteria (especially strain CL-1)-enhanced approach to reduce metal uptake of potato tubers in metal-polluted soils.

Bacteria from the Midgut of Common Cockchafer (Melolontha melolontha L.) Larvae Exhibiting Antagonistic Activity Against Bacterial Symbionts of Entomopathogenic Nematodes: Isolation and Molecular Identification.

The mechanisms of action of the complex including entomopathogenic nematodes of the genera Steinernema and Heterorhabditis and their mutualistic partners, i.e., bacteria Xenorhabdus and Photorhabdus, have been well explained, and the nematodes have been commercialized as biological control agents against many soil insect pests. However, little is known regarding the nature of the relationships between these bacteria and the gut microbiota of infected insects. In the present study, 900 bacterial isolates that were obtained from the midgut samples of Melolontha melolontha larvae were screened for their antagonistic activity against the selected species of the genera Xenorhabdus and Photorhabdus. Twelve strains exhibited significant antibacterial activity in the applied tests. They were identified based on 16S rRNA and rpoB, rpoD, or recA gene sequences as Pseudomonas chlororaphis, Citrobacter murliniae, Acinetobacter calcoaceticus, Chryseobacterium lathyri, Chryseobacterium sp., Serratia liquefaciens, and Serratia sp. The culture filtrate of the isolate P. chlororaphis MMC3 L3 04 exerted the strongest inhibitory effect on the tested bacteria. The results of the preliminary study that are presented here, which focused on interactions between the insect gut microbiota and mutualistic bacteria of entomopathogenic nematodes, show that bacteria inhabiting the gut of insects might play a key role in insect resistance to entomopathogenic nematode pressure.

Bacterial survival in whole blood depends on plasma sensitivity and resistance to neutrophil killing.

BACKGROUND: Whole blood (WB) is held at room temperature for not more than 24 hours before blood component manufacturing. The ability of several culture collection, skin-derived, and transfusion-related bacteria to survive in WB stored at 22 ± 2°C for 24 hours was investigated in this study. STUDY DESIGN AND METHODS: Twenty-one bacteria of the species Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus capitis, Streptococcus agalactiae, Serratia liquefaciens, Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, and Yersinia enterocolitica were inoculated into 7-mL aliquots of WB at a concentration of 500 colony-forming units (CFU)/mL. Spiked WB was stored aerobically at 22 ± 2°C, and bacterial viability and growth were monitored at 3, 8, and 24 hours during WB storage. Bacteria that showed decreased viability during WB incubation were further characterized for their sensitivity to plasma factors and neutrophil killing. RESULTS: There were three different scenarios for bacterial behavior during the hold of WB at 22 ± 2°C. Five bacteria proliferated (p < 0.03), 11 remained viable or showed low proliferation, and a third group of five bacteria had decreased or lost viability (p < 0.01). Three of the latter five bacteria were plasma-sensitive while the other two were plasma-resistant but susceptible to neutrophil killing (p = 0.01). CONCLUSIONS: The bactericidal activity of WB can be the result of plasma sensitivity or neutrophil killing. Bacteria with a starting inoculum of 500 CFU/mL, and able to resist WB immune factors, can proliferate to clinically significant levels posing a potential safety risk to transfusion patients. Results of this pilot study should be validated under standard WB collection and storage conditions.

Extending the 30-minute rule for red cell units - investigation of the bacterial risk of 60-minute exposures to ambient temperature.

BACKGROUND AND OBJECTIVES: In the UK, a significant proportion of red cell units is discarded due to the 30-min rule governing out of temperature control. Studies have shown that repeated warming to ambient temperature has little impact on red cell quality or bacterial growth. We aimed to validate extension of the rule to 60 minutes by investigation of repeated same, and different, day exposures on bacterial growth. MATERIALS AND METHODS: Red cell units were seeded individually at 100-1000 cfu/ml with Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas putida, Staphylococcus epidermidis, Enterobacter cloacae and Bacillus cereus. Test units were exposed to 30°C for 30 or 60 min on a single occasion at days 15, 17 and 21, or thrice on day 15 of a 35-day storage period. A 10-fold increase in bacterial counts in tests versus controls maintained in cold storage was considered indicative of significant bacterial proliferation. RESULTS: Exposure of units to 30°C for up to 60 min had no substantial impact on the growth of bacteria and all mesophiles declined steadily in tests and controls. Only P. putida showed a near significant elevation in count on exposure for 60 min at day 35. CONCLUSIONS: Extension of the out of temperature rule for red cells to 60 min will potentially not compromise patient safety, although exposures to ambient temperatures should be minimized. Units returned to storage must not be reissued for at least 6 hours and not be exposed to ambient temperatures on more than three occasions.

Metal-immobilizing Serratia liquefaciens CL-1 and Bacillus thuringiensis X30 increase biomass and reduce heavy metal accumulation of radish under field conditions.

In this study, metal-tolerant bacteria Serratia liquefaciens CL-1 and Bacillus thuringiensis X30 were compared for their Cd and Pb immobilization in solution and impacts on biomass and Cd and Pb uptake in a radish in metal-contaminated soils under field conditions. Strains CL-1 and X30 significantly reduced water-soluble Cd and Pb concentrations (45-67%) and increased the pH in solution compared to the controls. These strains significantly increased the biomass (25-99%) and decreased edible tissue Cd and Pb uptake in the radish (37-81%) and DTPA-extractable Cd and Pb contents (18-44%) of the rhizosphere soil compared to the un-inoculated controls. Strain CL-1 had higher potential to reduce edible tissue Cd and Pb uptake in the radish and DTPA-extractable Cd content than strain X30. Also, these strains significantly increased Cd translocation factor and strain CL-1 also significantly increased Pb translocation factor of the radish. Furthermore, strain CL-1 significantly increased the ratio of small soil aggregates (< 0.25 mm and 0.25-0.50 mm) of the rhizosphere soil. The results showed that these strains reduced the edible tissue Cd and Pb uptake through decreasing Cd and Pb availability in the soil and increasing Cd or Pb translocation from the roots to the leaves of the radish. The results also suggested the bacteria-related differences in reduced heavy metal uptake in the radish and the mechanisms involved under field conditions.

Serratia liquefaciens Causing Severe Ocular Damage in Noncontact Lens Wearer.

Serratia liquefaciens is a rarely encountered gram-negative organism in ophthalmology practice. The only reported ocular infections are from contamination of contact lenses. The authors report the first case of a patient who developed orbital cellulitis secondary to severe S. liquefaciens microbial keratitis.

Serratia liquefaciens KM4 Improves Salt Stress Tolerance in Maize by Regulating Redox Potential, Ion Homeostasis, Leaf Gas Exchange and Stress-Related Gene Expression.

High salinity mitigates crop productivity and quality. Plant growth-promoting soil rhizobacteria (PGPR) improve plant growth and abiotic stress tolerance via mediating various physiological and molecular mechanisms. This study investigated the effects of the PGPR strain Serratia liquefaciens KM4 on the growth and physiological and molecular responsiveness of maize (Zea mays L.) plants under salinity stress (0, 80, and 160 mM NaCl). High salinity significantly reduced plant growth and biomass production, nutrient uptake, leaf relative water content, pigment content, leaf gas exchange attributes, and total flavonoid and phenolic contents in maize. However, osmolyte content (e.g., soluble proteins, proline, and free amino acids), oxidative stress markers, and enzymatic and non-enzymatic antioxidants levels were increased in maize under high salinity. On the other hand, Serratia liquefaciens KM4 inoculation significantly reduced oxidative stress markers, but increased the maize growth and biomass production along with better leaf gas exchange, osmoregulation, antioxidant defense systems, and nutrient uptake under salt stress. Moreover, it was found that all these improvements were accompanied with the upregulation of stress-related genes (APX, CAT, SOD, RBCS, RBCL, H⁺-PPase, HKT1, and NHX1), and downregulation of the key gene in ABA biosynthesis (NCED). Taken together, the results demonstrate the beneficial role of Serratia liquefaciens KM4 in improving plant growth and salt stress tolerance in maize by regulating ion homeostasis, redox potential, leaf gas exchange, and stress-related genes expression.

First Pediatric Case of Infective Endocarditis Caused by Serratia Liquefaciens.

Infective endocarditis (IE) caused by Serratia liquefaciens has been reported in only 2 adults. We experienced the first pediatric (neonatal) case of IE caused by S. liquefaciens, with mitral valve vegetation 4 days after a palliative heart surgery. This report underscores the importance of treating for both gram-positive and gram-negative bacteria in IE cases until the blood cultures elucidate the details.

Bacteria can proliferate in thawed cryoprecipitate stored at room temperature for longer than 4 h.

Although key coagulation factor activities are maintained in thawed cryoprecipitate stored for up to 24 h at ambient temperature, several jurisdictions limit such storage to 4-6 h. Here, we separately spiked thawed cryoprecipitate units with four bacterial strains: Staphylococcus epidermidis, Serratia liquefaciens, Pseudomonas putida and Pseudomonas aeruginosa. No strains grew in the first 4 h of storage, but by 24 h, three of four exhibited up to 1000-fold proliferation. Pathogen inactivation technologies could be explored to mitigate the safety risk posed by extending storage of thawed cryoprecipitate at room temperature.

Evaluation of the spoilage potential of bacteria isolated from chilled chicken in vitro and in situ.

Microorganisms play an important role in the spoilage of chilled chicken. In this study, a total of 53 isolates, belonging to 7 species of 3 genera, were isolated using a selective medium based on the capacity to spoil chicken juice. Four isolates, namely Aeromonas salmonicida 35, Pseudomonas fluorescens H5, Pseudomonas fragi H8 and Serratia liquefaciens 17, were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ. The in vitro studies showed that A. salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins. However, the major spoilage isolate in situ was P. fragi H8, which exhibited a fast growth rate, slime formation and increased pH and total volatile basic nitrogen (TVBN) on chicken breast fillets. The relative amounts of volatile organic compounds (VOCs) originating from the microorganisms, including alcohols, aldehydes, ketones and several sulfur compounds, increased during storage. In sum, this study demonstrated the characteristics of 4 potential spoilage bacteria on chilled yellow-feather chicken and provides a simple and convenient method to assess spoilage bacteria during quality management.

Molecular detection by analysis of the 16S rRNA gene of fecal coliform bacteria from the two Korean Apodemus species (Apodemus agrarius and A. peninsulae).

Wild mouse feces can disseminate zoonotic microorganisms throughout a farm, which is a great threat to human health and can lead to economic loss through contaminated agricultural produce. To assess the microbial communities, especially fecal coliform bacteria, we used two methods. First, we isolated bacterial colonies onto the common media LB (lactose broth) agar, TSA (tryptic soy agar), and MRS (de Man, Rogosa, and Sharpe) agar, and then randomly select colonies from each plate and stocked them to the mother plate for genomic DNA isolation. Second, we analyzed bacterial colonies using the 16S rRNA gene molecular diagnostic method. Based on bacterial cultures and bacterial 16S rRNA gene markers, we detected four different bacterial species (Bacillus amyloliquefaciens, Escherichia coli, Staphylococcus xylosus, and Serratia liquefaciens) from fecal coliforms of the striped field mouse Apodemus agrarius and A. peninsulae in agricultural areas in South Korea. These results could help us to better understand the pathogen reservoirs of mice and initiate some preventive measures to mitigate the microbial risks associated with mouse fecal matter in agricultural production areas.

Ammonia-Oligotrophic and Diazotrophic Heavy Metal-Resistant Serratia liquefaciens Strains from Pioneer Plants and Mine Tailings.

Mine tailings are man-made environments characterized by low levels of organic carbon and assimilable nitrogen, as well as moderate concentrations of heavy metals. For the introduction of nitrogen into these environments, a key role is played by ammonia-oligotrophic/diazotrophic heavy metal-resistant guilds. In mine tailings from Zacatecas, Mexico, Serratia liquefaciens was the dominant heterotrophic culturable species isolated in N-free media from bulk mine tailings as well as the rhizosphere, roots, and aerial parts of pioneer plants. S. liquefaciens strains proved to be a meta-population with high intraspecific genetic diversity and a potential to respond to these extreme conditions. The phenotypic and genotypic features of these strains reveal the potential adaptation of S. liquefaciens to oligotrophic and nitrogen-limited mine tailings with high concentrations of heavy metals. These features include ammonia-oligotrophic growth, nitrogen fixation, siderophore and indoleacetic acid production, phosphate solubilization, biofilm formation, moderate tolerance to heavy metals under conditions of diverse nitrogen availability, and the presence of zntA, amtB, and nifH genes. The acetylene reduction assay suggests low nitrogen-fixing activity. The nifH gene was harbored in a plasmid of ∼60 kb and probably was acquired by a horizontal gene transfer event from Klebsiella variicola.

Isolation of bacterial strains able to metabolize lignin and lignin-related compounds.

UNLABELLED: In this study, we identified five strains isolated from soil and sediments able to degrade kraft lignin, aromatic dyes and lignin derivatives. Using 16S rRNA gene sequencing, the isolates were identified as Serratia sp. JHT01, Serratia liquefacien PT01, Pseudomonas chlororaphis PT02, Stenotrophomonas maltophilia PT03 and Mesorhizobium sp. PT04. All the isolates showed significant growth on lignin with no water-extractable compounds. Synthetic aromatic dyes were used to assess the presence of oxidative enzymes. All the isolates were able to use the thiazine dye Methylene blue and the anthraquinone dye Remazol Brilliant Blue R as the sole carbon source. Guaiacol, veratryl alcohol and biphenyl were also mineralized by all the strains isolated. These results suggest they could be used for the treatment of aromatic pollutants and for the degradation of the lignocellulosic biomass. SIGNIFICANCE AND IMPACT OF THE STUDY: The valorization of waste lignin and lignocellulosic biomass by biocatalysis opens up new possibilities for the production of value-added substituted aromatics, biofuel and for the treatment of aromatic pollutants. Bacteria with ligninolytic potential could be a source of novel enzymes for controlled lignin depolymerization. In this work, five soil bacteria were isolated and studied. Every isolate showed significant growth on lignin and was able to degrade several lignin monomers and ligninolytic indicator dyes. They could thus be a source of novel ligninolytic enzymes as well as candidates for a bacterial consortium for the delignification of lignocellulosic biomass.