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Our method was implemented in the Google Earth Engine (GEE) platform19. We divided it into four steps. Figure 2 summarizes our approach, including the input of the raw data (land-use and land-cover from 1985 to 2018 and the water surface), and the output data (from 1986 to 2018), which included maps of the annual secondary forest increment (Product 1), annual secondary forest extent (Product 2), annual secondary forest loss (Product 3; from 1987 to 2018), and annual secondary forest age maps (Product 4).
Fig. 2

Workflow of the proposed method.

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Input data
We used the land-use and land-cover data from the Brazilian Annual Land-Use and Land-Cover Mapping Project (MapBiomas Collection 4.1; https://mapbiomas.org/en/colecoes-mapbiomas-1)1 as input data. This dataset was obtained through the classification of images from the Landsat satellite series (30-m spatial resolution) using a theoretical algorithm implemented in the GEE platform19. Details about the processing of the dataset can be found in the Algorithm Theoretical Basis Document20. More detail about the land-use and land-cover classes can be found in the MapBiomas website (https://mapbiomas.org/en/codigos-de-legenda?cama_set_language=en).
Moreover, we used the maximum water surface extent data (from 1984 to 2018) developed by Pekel et al.21 (https://global-surface-water.appspot.com) to avoid the inclusion of false detection within wetland areas in our products. This dataset contains a map of the spatial distribution of the water surface cover from 1984 to 2018, globally21. These data were obtained from 3,865,618 Landsat 5, 7, and 8 scenes acquired between 16 March 1984 and 31 December 2018. Each pixel was individually classified into water or non-water cover using an expert system21 implemented in the GEE platform19.
Step 1 – Reclassifying MapBiomas data
All MapBiomas land-use and land-cover maps from 1985 to 2018 (34 maps) were reclassified into binary maps. We assigned the value “1” for all pixels in the Forest formation class of the MapBiomas product (Legend ID: 3) and “0” for the other land-use and land-cover classes. In our reclassified maps, pixels with value of “1” were, then, associated to the class “Forest”, which includes only forests classified as old-growth and secondary (before 1985). Mangrove and forest plantation classes were excluded from our secondary forest map.
Step 2 – Mapping the Annual Increment of Secondary Forests
We mapped the annual increment of secondary forests using the forest maps produced in Step 1. This process was carried out pixel-by-pixel, where every pixel classified as Forest (value 1) in the analysed year (yi; between 1986 to 2018) and classified as non-forest (value 0) in the previous year (yi-1; i = 1985, 1986… 2017) was mapped as secondary forest. As forest cover maps before 1985 were not available in the MapBiomas product, maps of secondary forest increment start in 1986, when it was possible to detect the first transition (1985 to 1986). Thus, 33 binary maps were obtained, where the secondary forest increments (non-forest to forest) have a value of 1 and the other transitions a value of 0 (forest to forest, non-forest to non-forest, and forest to non-forest). Here, we only considered secondary forest growth in pixels that had previously an anthropic cover (forest plantation, pasture, agriculture, mosaic of agriculture and pasture, urban infrastructure, and mining) and did not overlap wetland areas.
Step 3 – Mapping the Annual Extent of Secondary Forests
We generated 33 maps of the annual extent of secondary forests. To produce the map of secondary forest extent in 1987, we summed the map of the total secondary forest extent in 1986, which is the same map as the secondary forest increment in 1986 from step 2, with the 1987 increment map, resulting in a map containing all secondary forest pixels from 1986 and 1987. Knowing that the sequential sum of these maps results in pixels with values higher than 1, to create annual binary maps of secondary forest extent, we reclassified the map produced for each year by assigning the value 1 to pixels with values between 2 and 33 (secondary forest extent) and pixels with a value 0 were kept unchanged. Finally, to remove all secondary forest pixels that were deforested in 1987, keeping in the map only pixels with the extent of stand secondary forests, we multiplied the resulting map by the annual forest cover map of 1987, produced in step 1 (Fig. 3). This procedure was applied year-by-year from 1986 to 2018 to produce the maps of annual secondary forest extent. The removal of deforested pixels provides a product depicting the extent of secondary forest deforested in each specific year and they were also included as complimentary maps (from 1987 to 2018) in our dataset.
Fig. 3

Conceptual model of the approach used to calculate the age of secondary forests throughout the Brazilian territory.

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Step 4 – Calculating the Age of Secondary Forest
Finally, we calculated the age of the secondary forests (Fig. 3). First, we summed the 1986 map of annual secondary forest extent (from Step 3) with the 1987 map to obtain the age of secondary forests in 1987 (Fig. 4). We continued this summation year-by-year until the secondary forest age map of 2018 was obtained (Fig. 4). The values of each pixel in 2018 correspond to the age of the secondary forest. To ensure the elimination of deforested secondary forests from each age map, we executed a similar procedure as described in step 3 by removing all forest pixels overlaying non-forest areas (Fig. 4). As our analyses started in 1986, it was not possible to identify secondary forests before this year. The 1986 age map, therefore, only shows one-year old secondary forests, and the 2018 map shows ages of secondary forest varying between 1 and 33 years (Fig. 4). If a secondary forest pixel with any age is cleared in a given year, it is then removed and a value of zero is attributed to the pixel. The age of this pixel, subsequently, will only be computed again if the algorithm detects a new non-forest to forest transition in the forest cover map (Step 1), which depends on the MapBiomas project classification method.
Fig. 4

(a) Scatter-plot for the relationship between the proportion of the secondary forest within the 10 by 10 km cells in the two datasets. The dashed blue line is the 1:1 line; the red line is the average regression from the bootstrap approach with 10,000 interactions; the dashed red lines are regressions using the standard deviation values of the equation parameters. All p-values from the 10,000 bootstrap interactions were lower than 0.001. (b) Jitter-plot for the proportion of the secondary forest within the 10 by 10 km cells. The red dot is the mean, and the red vertical line the standard deviation.

Full size image More

Affiliations

The Intergovernmental Authority on Development Climate Prediction and Applications Centre (ICPAC), Nairobi, Kenya
Abubakr A. M. Salih, Marta Baraibar, Kenneth Kemucie Mwangi & Guleid Artan

Authors
Abubakr A. M. Salih

Marta Baraibar

Kenneth Kemucie Mwangi

Guleid Artan

Corresponding author
Correspondence to Abubakr A. M. Salih. More

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Microorganisms and medium composition
C. acetobutylicum DSM 792 and E. aerogenes DSM 30053 were used for all experiments. A modified Clostridium-specific medium without yeast extract was used for growth of mono-culture C. acetobutylicum as previously described in detail elsewhere71. The medium was prepared containing (per L): 0.5 g of KH2PO4, 0.5 g of K2HPO4 and 2.2 g of NH4CH3COO and glucose or cellobiose were added at a concentration of 999 C-mmol. The pH was arranged with 1 mol L−1 NaOH to 6.8. Trace elements solution was prepared as stock 100× solution containing (per L): 0.2 g of MgSO4·7 H2O, 0.01 g of MnSO4·7H2O, 0.01 g of FeSO4·7H2O, 0.01 g of NaCl. Vitamin solution was prepared as stock 200× solution containing (per L): 0.9 g of thiamine, 0.002 g of biotin and 0.2 g of 4-aminobenzoic acid. The trace elements solution and the vitamin solution were used for all experiments. Mono-culture of E. aerogenes was grown in a defined Enterobacter-specific medium, as described elsewhere72. The Enterobacter-specific medium was prepared containing (per L): 13.3 g K2HPO4, 4 g (NH4)2HPO4, 8 mg EDTA and trace elements (2.5 mg CoCl2·6H2O, 15 mg MnCl2·4H2O, 1.5 g CuCl2·4H2O; 3 mg H3BO3; 2.5 mg Na2MoO4·2H2O, 13 mg of Zn(CH3COO)2·2H2O). Glucose and cellobiose were prepared as stock solutions. Media, trace element solution, glucose and cellobiose solutions were flushed with sterile N2 to make the solutions anaerobic and sterilized separately at 121 °C for 20 min. Sterile anaerobic solutions of glucose or cellobiose, trace elements solution and filter sterilized vitamin solution were added into the media before the inoculation inside the sterilized biological safety cabinet (BH-EN 2005, Faster Srl, Ferrara, Italy).
Design of experiments
A mutual medium accommodating the nutritional requirements of both organisms was designed by using the DoE approach. The buffer compositions of two species specific media described above were analysed and the optimum concentrations of AC (NH4Cl), SA (Na+ acetate) and PB (KH2PO4/K2HPO4) capacity were investigated. The setting of DoE for concentration effect of AC, SA and PB capacity was based on 29 randomized runs within concentration range from 3–30 mmol L−1 of AC, 3–150 mmol L−1 of KH2PO4 and 10–120 mmol L−1 of SA (Table 1). Each experiment was performed in triplicates (n = 3), except for set E of the DoE experiment (centre points), which were performed in pentaplicate (n = 5). The DoE experiments were performed twice (N = 2). The end of the exponential growth phase of E. aerogenes and C. acetobutylicum was reached at 45 and 51.5 h, respectively. For modelling, these time points were used. The reason for providing an acetate source in the medium was due to the possibility to add an acetate oxidizing microorganism to the co-culture consortium, which was not performed in the context of this study.
Closed batch cultivations
Cultures of E. aerogenes and C. acetobutylicum were grown anaerobically at 0.3 bar in a 100 Vol.-% N2 atmosphere in a closed batch set-up33. Mono-culture and consortium closed batch experiments were conducted with the final volume of 50 mL medium in 120 mL serum bottles (Ochs Glasgerätebau, Langerwehe, Germany). Each serum bottle contained 45 mL Clostridium-specific medium, Enterobacter-specific medium or E-medium, 0.25 mL vitamin solution, 3.0 mL glucose or cellobiose stock solution, 0.5 mL trace elements solution and 1.25 mL inoculum. The serum bottles were sealed with rubber stoppers (20 mm butyl ruber, Chemglass Life Science LLC, Vineland, USA). For consortium experiments, different inoculum ratios were tested and initial cell concentrations were arranged with the ratios of (E. aerogenes : C. acetobutylicum) 1:2, 1:10, 1:100, 1:1000, 1:10,000 and 1:100,000 at a temperature of 37 °C. Pre-culture of E. aerogenes was diluted in DoE E-medium (Table 1) to inoculate the organism at cell densities of aforementioned ratios. The pressure in the headspace of the serum bottles were measured individually using a manometer (digital manometer LEO1-Ei,−1…3 bar, Keller, Germany). After each measurement, the pressure was released completely from the headspace of serum bottle by penetrating the butyl rubber stopper with a sterile needle. The pressure values were added up to reveal total produced pressure (cumulative pressure). Experiments were performed three times (N = 3) and each set was performed in quadruplicates (n = 4).
Cell counting, absorption measurements, DNA extraction and qPCR
A volume of 1 mL of liquid sample was collected by using sterile syringes at regular intervals for monitoring biomass growth by measuring the absorbance (optical density at 600 nm (OD600)) using a spectrophotometer (Beckman Coulter Fullerton, CA, USA). Every sampling operation was done inside the sterilized biological safety cabinet (BH-EN 2005, Faster Srl, Ferrara, Italy).
E. aerogenes and C. acetobutylicum cells were counted using a Nikon Eclipse 50i microscope (Nikon, Amsterdam, Netherlands) at each liquid/biomass sampling point. The samples for cell count were taken from each individual closed batch run using syringes (Soft-Ject, Henke Sass Wolf, Tuttlingen, Germany) and hypodermic needles (Sterican size 14, B. Braun, Melsungen, Germany). Ten microlitres of sample were applied onto a Neubauer improved cell counting chamber (Superior Marienfeld, Lauda-Königshofen, Germany) with a grid depth of 0.1 mm.
DNA for qPCR was extracted from 1 mL culture samples by centrifugation at 4 °C and 13,400 r.p.m. for 30 min. The following steps were applied for DNA extraction; (1) cells were resuspended in pre-warmed (65 °C) 1% sodium dodecyl sulfate (SDS) extraction buffer and (2) transferred to Lysing Matrix E tubes (MP Biomedicals, Santa Ana, CA, USA) containing an equal volume of phenol/chloroform/isoamylalcohol (25:24:1). (3) Cell lysis was performed in a FastPrep-24 (MP Biomedicals, NY, USA) device with speed setting 4 for 30 s and the lysate was centrifuged at 13,400 r.p.m. for 10 min. (4) An equal volume of chloroform/isoamylalcohol (24:1) was added to the supernatant of the lysate, followed by centrifugation at 13,400 r.p.m. for 10 min and collection of the aqueous phase. (5) Nucleic acids were precipitated with double volume of polyethylenglycol (PEG) solution (30% PEG, 1.6 mol L−1 NaCl) and 1 μL glycogen (20 mg mL−1) as carrier, incubated for 2 h at room temperature. (6) Following centrifugation at 13,400 r.p.m. for 1 h, nucleic acid pellets were washed with 1 mL cold 70% ethanol, dried at 30 °C using a SpeedVac centrifuge (Thermo Scientific, Dreieich, Germany), eluted in Tris-EDTA buffer and stored at −20 °C until further analysis. Nucleic acid quantification was performed with NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). qPCR assays were developed for quantifying E. aerogenes and C. acetobutylicum in consortium. The primer pairs were designed by targeting species specific genes (Supplementary Table 6) to prevent false-positive amplification and sequences of genes were compared for identifying optimal primer using the ClustalW2 multiple sequence alignment programme (http://www.ebi.ac.uk/Tools/clustalw2/). qPCR assays were performed in Eppendorf Mastercycler epgradientS realplex2 (Eppendorf, Hamburg, Germany). The PCR mixture (20 μL) contained 10 μL SYBR Green labelled Luna Universal qPCR Master Mix (M3003L, New England Biolabs), 0.5 μL of forward and 0.5 μL reverse primer, 8 μL sterile DEPC water and 1 μL of DNA template. Negative controls containing sterile diethyl pyrocarbonate (DEPC) water as a replacement for the DNA templates and DNA template of the non-targeted species were included separately in each run. The amplification protocol started with an initial denaturation at 95 °C for 2 min, followed by 45 cycles of denaturation at 95 °C for 30 s, annealing and fluorescence acquisition at 60 °C for 30 s and elongation at 72 °C for 30 s. A melting-curve analysis (from 60 °C to 95 °C at a transition rate of 1 °C every 10 s) was performed to determine the specificity of the amplification. All amplification reactions were performed in triplicates. A standard curve was generated as described elsewhere29. Culture samples of each organism were collected at different time intervals for cell count and genomic DNA extraction cell density of each strain were determined by cell counting under microscope during growth and subsequent gDNA extraction was applied to reflect absolute quantification. Six tenfold dilution standards were prepared and a linear regression analysis was performed between qPCR reads and cell counts and OD600 measurements.
Quantification of gas composition
Gas chromatography (GC) measurements were performed from serum bottles that remained without any manipulation after inoculation until the first time point GC measurement. After every GC measurement, remaining gas was released completely from the serum bottles by penetrating the butyl rubber stopper using a sterile needle. The pressure of serum bottles headspace was determined to examine whether there was any remaining overpressure by using a manometer (digital manometer LEO1-Ei,−1…3 bar, Keller, Germany). The gas compositions were analysed by using a GC (7890 A GC System, Agilent Technologies, Santa Clara, USA) with a 19808 Shin Carbon ST Micropacked Column (Restek GmbH, Bad Homburg, Germany) and provided with a gas injection and control unit (Joint Analytical System GmbH, Moers, Germany) as described before73,74,75. The standard test gas employed in GC comprised the following composition: 0.01 Vol.-% CH4; 0.08 Vol.-% CO2 in N2 (Messer GmbH, Wien, Austria). All chemicals were of highest grade available. H2, CO2, N2, 20 Vol.-% H2 in CO2 and 20 Vol.-% CO2 in N2 were of test gas quality (Air Liquide, Schwechat, Austria).
Quantification of liquid metabolites
Quantification of sugars, volatile fatty acids and alcohols were performed with high-performance liquid chromatography (HPLC) system (Agilent 1100), consisting of a G1310A isocratic pump, a G1313A ALS autosampler, a Transgenomic ICSep ICE-ION-300 column, a G1316A column thermostat set at 45 °C and a G1362A RID refractive index detector, measuring at 45 °C (all modules were from Agilent 1100 (Agilent Technologies, CA, USA). The measurement was performed with 0.005 mol L−1 H2SO4 as solvent, with a flow rate of 0.325 mL min−1 and a pressure of 48–49 bar. The injection volume was 40 µL.
Data analysis
For the quantitative analysis, the maximum specific growth rate (µmax [h−1]) and mean specific growth rate (µmean [h−1]) were calculated as follows: N = N0·eµt with N, cell number [cells ml−1]; N0, initial cell number [cells ml−1]; t, time [h] and e, Euler’s number. According to the delta cell counts in between sample points, µ was assessed. The Y(H2/S) [mol mol−1], HER [mmol L−1 h−1], CER [mmol L−1 h−1] and the specific H2 production rate (qH2) [mmol g−1 h−1]32 were calculated from the intervals between each time point and the gas composition in the headspace of serum bottle was determined using the GC. The elementary composition of the corresponding biomass59 was used for the calculation of the mean molar weight, carbon balance and the DoR balance. Yields of byproducts were determined after HPLC measurement. Values were normalized according to the zero control. Moreover, the Shannon diversity index (H) was calculated to interpret the changes in microbial diversity, accounting for both richness (S), the number of species present and abundance of different species. Relative abundance of two species was evaluated according to the calculated evenness (EH) values76. Global substrate uptake rate, byproduct production rates and the mass balance analyses of the mono-cultures and consortium on glucose and cellobiose were calculated between the first and last time point.
Fluorescence in situ hybridization
For FISH, samples of 2 mL were collected for cell fixation. The samples were centrifuged in micro-centrifuge (5415-R, Eppendorf, Hamburg, Germany) for 10 min at 13,200 r.p.m. and pellets were resuspended in 0.5 mL phosphate-buffered saline (PBS) (10 mmol L−1 of Na2HPO4/NaH2PO, 130 mmol L−1 of NaCl, pH of 7.2–7.4). After repeating this procedure twice, 0.5 mL ice-cold absolute ethanol was added to the 0.5 mL PBS/cell mixture. The ethanol fixed samples were thoroughly mixed and then stored at −20 °C. Poly-l-lysine solution (0.01 % (v/v)) was used for coating the microscope slides (76 × 26 × 1 mm, Marienfeld-Superior, Lauda-Königshofen, Germany) containing ten reaction wells separated by an epoxy layer. After dipping the slide into the solution for 5 min, residual poly-l-lysine from the slides was removed by draining the well, followed by air-drying for several minutes. Cells were immobilized on prepared slides by adding samples (1–10 µL) on each well and air-drying. For cell dehydration, the slides were impregnated with ethanol concentrations of 50% (v/v), 80% (v/v) and 96% (v/v), respectively. The slides were dipped into each solution for 3 min, starting from the lowest concentration.
The EUB338 probe77 was used to target specific 16S rRNA found in almost all organisms belonging to the domain of bacteria78. The GAM42a probe specifically binds to target regions of gammaproteobacterial 23S rRNA79 (Supplementary Table 7). Both probes were diluted with DEPC water to a certain extent depending on the fluorescence label. Cy3-labelled EUB338 was diluted to a probe concentration of 30 ng DNA μL−1, whereas FLUOS-labelled GAM42a was adjusted to a final concentration of 50 ng DNA μL−1. For hybridization of the probe, 20 µL of hybridization buffer (900 mmol L−1 NaCl, 20 mmol L−1 Tris/HCl, 30% formamide (v/v), 0.01% SDS (v/v)) and 2 µL of diluted probe solution were added into each well. The hybridization reaction (46 °C, overnight) was facilitated using an airtight hybridization chamber (50 mL centrifuge tube) to prevent dehydration.
A stringent washing step was performed at 48 °C for 10 min in pre-warmed 50 mL washing buffer (100 mmol L−1 NaCl, 20 mmol L−1 Tris/HCl, 5 mmol L−1 EDTA). Afterwards, the slides were dried up and a mounting medium (Antifade Mounting Medium, Vectashield Vector Laboratories, CA, USA) was added to each well. The slides were sealed with a cover glass and examined under phase-contrast microscope (Nikon Eclipse Ni equipped with Lumen 200 Fluorescence Illumination Systems) using filter sets TRITC (557/576) (maximum excitation/emission in nm) for cy3-labelled EUB338 probe and FITC (490/525) for FLUOS-labelled GAM42a probes by a 100 × 1.45 numerical aperture microscope objective (CFI Plan Apo Lambda DM ×100 Oil; Nikon Corp., Japan).
Statistics and reproducibility
DoE experiments were designed and analysed using Design Expert version 11.1.2.0 (Stat-Ease, Inc. USA). Analysis of variation was performed at α = 0.05. The p-values for each test are indicated in the ‘Results’ section. All closed batch experiments were reproduced three times (N = 3) and each replication contained quadruplicate (n = 4). qPCR and FISH experiments, which applied all of the mentioned replicates, were performed in technical triplicates (n = 3). DoE experiments were conducted twice (N = 2) and each replication contained triplicate experiments for corner points (n = 3), except the set E (centre points), which was performed in biological pentaplicates (n = 5).
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article. More

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