Co-cultivation of Isochrysis galbana

and Marinobacter sp. can enhance algal

growth and docosahexaenoic acid
production Ying-Ying Wang
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 Aquaculture 556 (2022) 738248

Contents lists available at ScienceDirect

Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Co-cultivation of Isochrysis galbana and Marinobacter sp. can enhance algal

growth and docosahexaenoic acid production
Ying-Ying Wang a, Si-Min Xu a, Jia-Yi Cao a, *, Min-Nan Wu a, Jing-Hao Lin a, Cheng-Xu Zhou a,
Lin Zhang a, Hai-Bo Zhou b, Yan-Rong Li d, Ji-Lin Xu a, *, Xiao-Jun Yan c
a
Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ministry of Education of China, Ningbo, Zhejiang 315211, China
b
Fujian Dalai Seed Science and Technology Co. LTD, Ningde, Fujian 352101, China
c
Collaborative Innovation Center for Zhejiang Marine High-Efficiency and Healthy Aquaculture, Ningbo University, Ningbo, Zhejiang 315211, China
d
Ningbo Institute of Oceanography, Ningbo, Zhejiang 315832, China

A R T I C L E I N F O A B S T R A C T

Keywords: Isochrysis galbana, an important diet microalgal species, is widely used in aquaculture and rich in docosahexa­
Isochrysis galbana enoic acid (DHA). Currently, no efficient strategies are available to steadily enhance the production of its biomass
Marinobacter sp. and DHA. It has been reported that algae-associated bacteria can affect the yield of algae and the production of
Growth-promoting bacteria
high-value compounds. Here, we identified a growth-promoting bacterial strain Marinobacter sp., which could
DHA
enhance the growth, the content of chlorophyll a, the maximal photochemical efficiency of PS II (Fv/Fm), and
soluble protein content of I. galbana when they were co-cultured, while superoxide dismutase (SOD) activity was
decreased. Besides, Marinobacter sp. promoted the production of DHA and up-regulated the expressions of genes
involved in DHA synthesis pathway of I. galbana. Our study clearly suggested that co-cultivation of I. galbana and
Marinobacter sp. could effectively enhance the quality and quantity of microalgae, showing promising applica­
tions in improving productivity and sustainability of aquaculture algal rearing systems.

1. Introduction cultivation of high-quality microalgae and improving their high-value

Microalgae have potential applications as food, medicine, health
products, biomass energy, industrial materials, bait in aquaculture and
so on (Harun et al., 2010; Hemaiswarya et al., 2011; Matos et al., 2017).
Microalgae are rich in nutrients, including proteins, amino acids, pig­
ments, carbohydrates, lipids, vitamins, and some minerals (Buono et al.,
2014; Matos et al., 2017; Pradhan et al., 2021). Among them, docosa­
hexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the most
valuable functional components in microalgal lipids, which have been
widely believed to benefit human health (Romieu et al., 2005). Besides,
these microalgal nutrients have also been widely used in aquaculture.
Microalgae as bait or bait supplements can alleviate the barriers to fish
meal farming and have been proved to be a successful replacement for
fish meal (Dineshbabu et al., 2019). Some microalgae, such as Tha­
lassiosira pseudonana, are widely cultivated to feed the Pacific oyster
Crassostrea gigas and rock scallops (Yaakob et al., 2014). Therefore, the
substances are very important for aquaculture.
As one of the most important bait microalgae, I. galbana belongs to
the class of Prymnesiophyceae (Matos et al., 2019). Extensive research
has shown that I. galbana is widely used to feed bivalve larvae, such as
mussels, scallops, clams, and bivalve larvae (Dineshbabu et al., 2019;
Tremblay et al., 2007), because it is small in size, easy to be digested,
and rich in essential nutritional constituents, such as DHA and EPA. As a
potential algal strain with abundant DHA and EPA, it has been observed
that I. galbana is not only a good bait for aquaculture seedling but also an
important raw material for the development of bioactive substances (Liu
et al., 2013). Meanwhile, it is considered to have the potential to achieve
industrialization. Therefore, a great deal of attention has been paid to
the study of I. galbana recently.
To improve the wide application of I. galbana, it is of great signifi­
cance to enhance its biomass yield. Studies have found that the growth
of I. galbana is affected by many abiotic factors, such as light,

Abbreviations: DHA, docosahexaenoic acid; SOD, superoxide dismutase; EPA, eicosapentaenoic acid; BCA, bicinchoninic acid; FAMEs, Fatty acid methyl esters;
RT-qPCR, Real-time quantitative PCR; rbcL, ribulose-1,5-bisphosphate carboxylase/oxygenase; d4FAD, delta-4 desaturase gene; ASE2, delta-9 elongase gene; SFAs,
saturated fatty acids; MUFAs, monounsaturated fatty acids; ROS, reactive oxygen species; VF, vibrioferrin.
* Corresponding authors.
E-mail addresses: caojiayi@nbu.edu.cn (J.-Y. Cao), xujilin@nbu.edu.cn (J.-L. Xu).

https://doi.org/10.1016/j.aquaculture.2022.738248
Received 14 December 2021; Received in revised form 21 March 2022; Accepted 8 April 2022
Available online 11 April 2022
0044-8486/© 2022 Elsevier B.V. All rights reserved.
Y.-Y. Wang et al.
temperature, salinity, CO2, and nutrients (Alkhamis and Qin, 2013; Cao
et al., 2020; Picardo et al., 2013; Sayegh and Montagnes, 2011; Zhang
et al., 2014). However, even if the main abiotic factors of the growth
environment of I. galbana are controlled under optimal conditions, there
are still many problems under large-scale outdoor conditions, reminding
us that the growth is also impaired by other factors in addition to the
above-mentioned abiotic factors. Algae will release a large number of
carbohydrates, amino acids, enzymes, lipids, and other metabolites into
the environment during the growth process to form a unique “phyco­
sphere”, which will attract a lot of bacteria-dominated microorganisms
(Bell and Mitchell, 1972). In this environment, the extracellular prod­
ucts of algae will stimulate bacterial growth and vice versa. Therefore,
the interactions between these two groups and the influence of their
interaction on each other are areas of recent research interest. Besides
the influence on growth, it has been reported that bacteria also affect the
production of high-value compounds of microalgae. For example,
Pseudomonas composti promoted the increase in biomass yield and lipid
of Characium sp. 46–4 by releasing some unidentified extracellular
compounds (Berthold et al., 2019). In addition, Liu et al. (2020) have
found that co-culture with probiotic algae-associated bacteria signifi­
cantly enhances the EPA production of Nannochloropsis oceanica. In the
case of I. galbana, Sandhya and Vijayan (2019) have shown that some
I. galbana-associated bacteria play a role in promoting algal growth.
Moreover, the interaction between them is regulated by various growth-
stimulating compounds, such as antioxidants, siderophores, and indole-
3-acetic acid, which can have a significant positive impact on algal
growth. However, the relationship between this microalgae and bacteria
remains largely unexplored. Therefore, it is urgently necessary to
explore the growth-promoting bacteria and their effects on the physi­
ology and metabolism of I. galbana, as well as the interaction mechanism
between them.
In the present study, we preliminarily applied different initial algae/
bacteria ratios to explore the effects of I. galbana-associated bacteria
Marinobacter sp. on the growth of I. galbana. We further evaluated the
physiological and biochemical effects of Marinobacter sp. on I. galbana.
Collectively, our current findings not only improved the understanding
of bacteria-microalgae interactions but also provided an alternative
strategy to improve algal production and culture stability.
2. Materials and methods
2.1. Culture of microalgae and bacteria
I. galbana 3011 was obtained from the Marine Biotechnology Labo­
ratory of Ningbo University, China. As a culture medium, the seawater
was filtered through 0.22-μm cellulose acetate membranes and then
sterilized by autoclaving. NMB3 medium used in this study was
composed of KNO3 (100 mg/L), KH2PO4 (10 mg/L), MnSO4⋅H2O (2.5
mg/L), FeSO4⋅7H2O (2.5 mg/L), EDTA-Na2 (10 mg/L), vitamin B1 (6 μg/
L), and vitamin B12 (0.05 μg/L) (Yang et al., 2016). A photoperiod of 12/
12-h day/night was applied with a light intensity of 100 μmol photon
m− 2 s− 1 using a cool light fluorescent lamp. The temperature was
controlled at 25 ◦ C throughout the experiment. Marinobacter sp. used in
this study was isolated from I. galbana culture and preserved in the
Marine Biotechnology Laboratory of Ningbo University. Marinobacter sp.
was freshly cultured on a 2216E marine medium at 28 ◦ C before
experiments.
2.2. Co-culture experiments
Before co-culture experiments, Marinobacter sp. was plated freshly on
a 2216E agar medium, and single colonies were grown in the 2216E
liquid medium overnight (25 ◦ C, 180 rpm). The freshly prepared bac­
terial culture broth was centrifuged at 5000 ×g for 5 min, washed twice,
and finally resuspended in sterile NMB3 medium. The axenic I. galbana
was obtained and maintained as previously described (Cao et al., 2019).
2
Aquaculture 556 (2022) 738248
When axenic I. galbana was cultured to an exponential phase (initial cell
density of about 1 × 106 cells/mL), the bacterial suspension was added
into the algal culture to achieve an algae/bacteria ratio of 1:1, 1:50, or
1:100 (cell counts: cell counts). Axenic I. galbana cultured alone was
denoted as the control group.
2.3. Physiological and biochemical determinations of microalgae
To study the physiological and biochemical effects of Marinobacter
sp. on I. galbana, the algae/bacteria ratio in the co-culture was 1:100.
The content of chlorophyll a, the maximal photochemical efficiency of
PS II (Fv/F), soluble protein content, and superoxide dismutase (SOD)
activity of I. galbana in the co-culture and mono-culture systems were
measured every 2 days. The content of chlorophyll a was measured as
previously described (Bruckner et al., 2008). The absorbance was
determined at wavelengths of 630, 664, and 750 nm. The content of
chlorophyll a was then calculated using the following equation:
Chl a (μg/mL) = 11.47 × (OD664 − OD750 ) − 0.4 × (OD630 − OD750 )
Fv/Fm was measured by AquaPen, which is a lightweight, hand-held
fluorometer intended for quick and reliable measurements of photo­
synthetic activity in algae. The measurement was performed with a
dark-adapted sample. Soluble protein was extracted and analyzed using
the bicinchoninic acid (BCA) protein assay with bovine serum albumin
as the standard according to the manufacturer’s instructions. The con­
centration of soluble protein was calculated based on the standard curve
drawn. The SOD activity was determined by the xanthine oxidase
method at a wavelength of 550 nm according to the manufacturer’s
instructions.
2.4. Fatty acid analysis
For fatty acid analysis, the samples from mono-culture of I. galbana,
mono-culture of Marinobacter sp., and their co-cultures were collected
on the 14th day post-co-culturing by centrifuging at 10,000 rpm for 10
min. The obtained I. galbana and Marinobancter sp. pellets were pre-
frozen in liquid nitrogen and dried by the vacuum freeze-drying sys­
tem for further analysis.
Fatty acid methyl esters (FAMEs) were purified following the
methods with modifications as previously described (Okuyama et al.,
1992). Briefly, samples (20 mg) of lyophilized cells were accurately
weighed and transferred to culture tubes. Next, 1 mL n-hexane, 15 μL
internal standard nonadecanoic acid (1 mg/mL), and 1.5 mL freshly
made pyromrcyl chloride (prepared by slowly adding 10 mL of acetyl
chloride to 100 mL of anhydrous methanol) were added to each sample
in a culture tube and vortexed for 1 min at a slow speed. The tightly
capped tubes were heated at 70 ◦ C in a water bath for 2 h. After samples
were cooled to room temperature, 2.5 mL of 6% K2CO3 was added,
followed by the addition of 1 mL n-hexane. The tubes were vortexed 30s
and the upper phase was collected in 2-mL sample bottle, then cen­
trifugated at 3000 rpm for 10 min. The supernatant was filtered by an
organic phase filter membrane, and the filtrate was collected into a 2-mL
screw-capped sample bottle. The purified fatty acids were subjected to
methylations.
The methyl esters were analyzed using an Agilent 7890B–7000C gas
chromatography system equipped with an EI detector. A 100-m-long
capillary column CD-2560 (Germany, CNW) was used. The column
had an inside diameter of 0.25 mm and a film thickness of 0.2 μm. The
temperature program was set as follows. The initial oven temperature
was maintained at 140 ◦ C for 5 min, then the temperature was increased
to 240 ◦ C at an increment of 4 ◦ C/min; and finally, the oven was
maintained isothermally for 30 min. The injector temperature was set at
250 ◦ C. Helium was used as the carrier gas at a constant flow rate of 2.25
mL/min. In each analysis, 1 μL of methyl ester solution was injected into
the chromatograph. Fatty acids were identified by comparison of
Y.-Y. Wang et al.
retention times to those of known standards and expressed as % of
FAMEs identified. The concentration of each fatty acid was calculated
using Qualitative Analysis, B.07.00 software.
2.5. Total RNA extraction
Microalgae were harvested through centrifugation at 10,000 rpm
and 4 ◦ C for 10 min. Algal cells were ground into powder in liquid ni­
trogen. Total RNA was isolated from each sample using an E. Z. N. A.®
Plant RNA Kit (OMEGA Bio-Tek) according to the manufacturer’s in­
structions. The concentration and quality of the purified RNA were
determined using a Thermo Scientific NanoDrop One spectrophotometer
(Thermo Fisher Scientific, USA).
2.6. Real-time quantitative PCR (RT-qPCR)
Briefly, purified RNA was reversely transcribed into cDNA using
PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time)
(TaKaRa, Japan). Gene expression was analyzed through RT-qPCR using
a LongGene Q2000A qPCR system (Hangzhou) and TB Greer® Premix
Ex Taq™ II (Tli RNaseH Plus) (TaKaRa, Japan). The large subunit gene
of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) was used as
the reference gene (Han et al., 2019). Primers for delta-4 desaturase
gene (d4FAD), d5FAD, d6FAD, and d8FAD were synthesized as previ­
ously reported (Huerlimann et al., 2014). The primers for the delta-9
elongase gene (ASE2) were designed using Primer 5. All the PCR
primers were listed in Table S1. The relative expressions of target genes
were calculated using the 2^-ΔΔCt method as previously described (Cao
et al., 2016).
3. Results
3.1. The effects of Marinobacter sp. on the growth of I. galbana
The axenic strain of I. galbana was cultured in the presence of Mar­
inobacter sp. to investigate the effect of Marinobacter sp. on microalgal
growth. Fig. 1 shows the effects of Marinobacter sp. with different algae/
bacteria ratios (1:1, 1:50, and 1:100) on the content of chlorophyll a of
I. galbana over a period of 14 days. When we explored the effects of
Marinobacter sp. on the growth of I. galbana, we found that Marinobacter
sp. exerted a growth-promoting effect on I. galbana depending on the
population density of the bacteria. Besides, the growth-promoting ef­
fects were enhanced with the increase of the treatment duration of
bacteria. The presence of bacteria under ratios of 1:50 and 1:100
significantly increased the cell growth of I. galbana compared with the
control group. We further detected the cell growth of these two strains
during the co-cultivation. The cell growth of I. galbana was enhanced by
Marinobacter sp., while the growth of Marinobacter sp. was decreased
when compared with pure culture of bacteria from day 4 (Fig. S1).
Moreover, no significant effect on the morphology of Marinobacter sp.
was found in co-cultivation. For I. galbana, at a later stage of the growth,
a large number of deformed algal cells were observed in the mono-
culture, while no deformation of algal cells was observed in the co-
cultures (Fig. S2).
3.2. The physiological and biochemical effects of Marinobacter sp. on
I. galbana
To study the physiological and biochemical effects of Marinobacter
sp. on I. galbana, the content of chlorophyll a, Fv/F, soluble protein
content, and SOD activity of I. galbana in the co-culture and mono-
culture systems were compared (Fig. 2). During 14 days of moni­
toring, the content of algal chlorophyll a in co-cultures was significantly
higher compared with the I. galbana mono-culture (Fig. 2A). The chlo­
rophyll fluorescence parameter, Fv/Fm, was used to monitor the effect of
Marinobacter sp. on the photosynthetic property of I. galbana. Fig. 2B
3
Aquaculture 556 (2022) 738248
Fig. 1. Co-culture results of Isochrysis galbana and Marinobacter sp. with three
different initial algae/bacteria ratios (1:1, 1:50, 1:100) expressed as chlorophyll
a content over the period of 14 days. Significance of the differences between
mean values was determined with Student’s t-test. All the experiments were
carried out in triplicate with standard error as error bar, while asterisks (*)
indicate significant difference at p value <0.05.
shows that Fv/Fm was higher in the co-culture system during the whole
culture process compared with the mono-culture. The results revealed
that the photosynthetic property of I. galbana was improved in the
presence of Marinobacter sp. The total concentration of soluble protein in
the microalgal cells was increased steadily over time (Fig. 2C), showing
that the presence of Marinobacter sp. significantly increased the syn­
thesis of soluble protein in I. galbana. The SOD activity in I. galbana was
decreased in the co-culture system compared with the mono-culture
(Fig. 2D). The presence of Marinobacter sp. inhibited the SOD activity
in microalgae during the entire study period. On the 8th day of the
experiment, the highest SOD activity was observed in the co-culture,
while it was still lower compared with the mono-culture at the corre­
sponding time point. The peak value in the mono-culture on the last day
was 1.27-fold higher compared with the co-culture group.
3.3. The effects of Marinobacter sp. on the fatty acid composition of
I. galbana
The difference in fatty acid composition of I. galbana in the co-culture
and mono-culture systems was analyzed (Table 1-3, Table S2). The most
abundant saturated fatty acids (SFAs) in both axenic and co-cultured
I. galbana were myristic acid (C14:0) and palmitic acid (C16:0). The
specific SFAs obtained from the mono-culture of Marinobacter sp. con­
sisted of dodecanoic acid (C12:0) and tridecanoic acid (C13:0). Addi­
tionally, the main SFAs in Marinobacter sp. were C12:0 (22.81 ± 0.66%)
and C16:0 (29.91 ± 1.03%). Moreover, we found that the contents of
pentadecanoic acid (C15:0), stearic acid (C18:0), and behenic acid
(C22:0) were significantly decreased in I. galbana when co-cultured with
Marinobacter sp. The levels of total monounsaturated fatty acids
(MUFAs) in axenic (27.75 ± 0.62%) and co-cultured (28.46 ± 0.28%)
I. galbana were similar. The most abundant MUFA in both axenic and co-
cultured I. galbana was elaidic acid (C18:1n-9 t). The specific MUFA
obtained from mono-cultured Marinobacter sp. was myristoleic acid
(C14:1n-5). Besides, the content of heptadecenoic acid (C17:1n-7) was
Y.-Y. Wang et al. Aquaculture 556 (2022) 738248

Fig. 2. Chlorophyll a content (A), Fv/Fm (B), protein content (C) and superoxide dismutase (SOD) activity (D) for Isochrysis galbana co-culture and mono-culture
during 14 d exposure time. Significance of the differences between mean values was determined with Student’s t-test. All the experiments were carried out in
triplicate with standard error as error bar, while asterisks (*) indicate significant difference at p value <0.05.

Table 1 Table 2
Saturated fatty acid composition (as a percentage of total fatty acids) of Iso­ Monounsaturated fatty acid composition (as a percentage of total fatty acids) of
chrysis galbana co-cultured with Marinobacter sp. for 14 days. Isochrysis galbana co-cultured with Marinobacter sp. for 14 days.
Saturated fatty Axenic I. galbana Axenic I. galbana Marinobacter sp. Monounsaturated Axenic I. galbana Axenic I. galbana Marinobacter sp.
acids (SFA’s) + Marinobacter sp. fatty acids (MUFA’s) + Marinobacter sp.

C12:0 ND ND 22.81 ± 0.66 C14:1n-5 ND ND 0.49 ± 0.02

C13:0 ND ND 1.10 ± 0.08 C16:1n-7 4.22 ± 0.16 5.15 ± 0.06* 7.47 ± 1.50
C14:0 8.9 ± 0.13 10.68 ± 0.07* 1.36 ± 0.16 C17:1n-7 1.13 ± 0.06 0.75 ± 0.01* 1.01 ± 0.18
C15:0 1.29 ± 0.09 0.87 ± 0.00* 0.32 ± 0.02 C18:1n-9 t 19.02 ± 0.22 18.68 ± 0.16 14.80 ± 0.22
C16:0 15.09 ± 0.37 14.55 ± 0.05 29.91 ± 1.03 C18:1n-9c 2.29 ± 0.09 2.62 ± 0.03* 3.55 ± 0.75
C18:0 2.24 ± 0.26 1.14 ± 0.20* 4.36 ± 0.30 C22:1n-9 1.09 ± 0.09 1.26 ± 0.02 ND
C20:0 0.73 ± 0.37 0.24 ± 0.00 ND ΣMUFA 27.75 ± 0.62 28.46 ± 0.28 27.32 ± 2.67
C22:0 2.16 ± 0.10 1.20 ± 0.02* ND
ΣSFA 30.41 ± 1.32 28.68 ± 0.34 59.86 ± 2.25 Asterisks (*) indicate significant difference at p value <0.05. ND = not detected.

Asterisks (*) indicate significant difference at p value <0.05. ND = not detected.

significantly decreased in co-cultured I. galbana. The levels of total
polyunsaturated fatty acids (PUFAs) were not significantly changed in
axenic I. galbana (40.38 ± 1.78%) compared with the co-cultures (42.05

4
Y.-Y. Wang et al. Aquaculture 556 (2022) 738248

Table 3 Therefore, it is necessary to find strategies to improve its production. In

Polyunsaturated fatty acid composition (as a percentage of total fatty acids) of our study, we identified a growth-promoting bacterial strain Mar­
Isochrysis galbana co-cultured with Marinobacter sp. for 14 days. inobacter sp. and DHA production of I. galbana can be enhanced by this
Polyunsaturated fatty Axenic Axenic I. galbana+ Marinobacter bacterium. As we known, this was the first study to find a bacterium that
acids (PUFA’s) I. galbana Marinobacter sp. sp. can both enhance the algal biomass and DHA production in I. galbana.
C16:2n-6 0.89 ± 0.08 1.13 ± 0.03* ND Our results lay a foundation for studying the interaction between algae
C18:2n-6 5.21 ± 0.54 3.47 ± 0.01 ND and bacteria and help improve the yield and quality of bait-microalgae
C18:3n-3 6.90 ± 0.10 6.07 ± 0.06* ND in aquaculture.
C18:4n-3 10.99 ± 13.91 ± 0.05* ND
When we explored the effects of Marinobacter sp. on the growth of
0.41
C20:2n-6 0.74 ± 0.07 0.39 ± 0.07* ND I. galbana, we found that the growth-promoting role of Marinobacter sp.
C20:3n-3 0.38 ± 0.04 0.21 ± 0.02* ND to I. galbana depends on the population density of the bacterium. With
C20:4n-6 0.33 ± 0.06 0.17 ± 0.00 ND the increase of bacterial density, the growth-promoting effects became
C20:4n-3 0.41 ± 0.08 0.15 ± 0.00* ND more obvious (Fig. 1). It is crucial to note that in cultures with a high
C20:5n-3 1.13 ± 0.12 1.16 ± 0.02 ND
C22:5n-6 2.22 ± 0.14 1.96 ± 0.03 ND
density of bacterial cells, the availability of light and nutrients to
C22:6n-3 11.18 ± 13.43 ± 0.10* ND microalgae cells is lower, and the growth rates and yields of valuable
0.14 compounds, such as carotenoids and fatty acids, decrease (Fuentes et al.,
ΣPUFA 40.38 ± 42.05 ± 0.39 ND 2016). Possibly Marinobacter sp. cell density in our study was not suf­
1.78
ficiently high to affect the availability of light and nutrients. Our results
Asterisks (*) indicate significant difference at p value <0.05. ND = not detected. also suggested that an appropriate algae/bacteria ratio was a crucial
factor to achieve high I. galbana growth in the co-cultivation system.
Similarly, the growth-promoting effect of Alteromonas macleodii on
± 0.39%). Our study clearly suggested that PUFAs were absent in I. galbana also depends on the population density of the bacteria (Cao
Marinobacter sp. The contents of hexadecadienoic acid (C16:2n-6), et al., 2021).
stearidonic acid (C18:4n-3), and DHA (C22:6n-3) in the co-cultured Our results also indicated in the presence of Marinobacter sp., the
I. galbana were significantly increased compared with the axenic content of chlorophyll a was always significantly higher than that of
I. galbana. However, the contents of α-linolenic acid (C18:3n-3), eico­ axenic algal culture (Fig. 2). This finding was consistent with a previous
sadienoic acid (C20:2n-6), eicosatrienoic acid (C20:3n-3) and eicosate­ study that Marinobacter sp. has positive effects on the growth of seven
traenoic acid (C20:4n-3) were significantly decreased compared with microalgal species (Ling et al., 2020). Besides, the co-cultivation of
the axenic I. galbana. Rhizobium sp. and Ankistrodesmus sp. results in an increment of up to
30% in chlorophyll (Do Nascimento et al., 2013). Likewise, a similar
3.4. The effects of Marinobacter sp. on the expressions of genes involved study has shown that the co-immobilization of Chlorella vulgaris and
in DHA synthesis pathway Azospirillum brasilense leads to increments of up to 35% in chlorophyll a
(Gonzalez and Bashan, 2000). The increase in the content of chlorophyll
ASE2, d4FAD, d5FAD, d6FAD, and d8FAD are key genes involved in a was indeed a manifestation of the algal growth promoted by Mar­
the addition of double bonds and carbon atoms to the growing fatty inobacter sp.
chain in the DHA synthesis pathway (Fig. S3). Based on RT-qPCR Stresses usually lead to the accumulation of reactive oxygen species
analysis, the expressions of these five genes were all up-regulated in (ROS). ROS are extremely harmful when their production exceeds the
co-cultures compared with the mono-cultures on all three time points (2 buffering capacity of the antioxidant system (Wang et al., 2019). It is
d, 4 d and 14 d after co-cultivation) (Fig. 3). Among them, the expression well known that SOD plays a key role in the removal of ROS. We found
of d8FAD was mostly enhanced (fold change = 12.99) on 14 d. that the SOD activity of I. galbana was always lower compared with the
control group during the co-culture period (Fig. 2). Similarly, co-culture
4. Discussion with A. macleodii severely inhibits the SOD activity of I. galbana
compared with the control group during the entire study period, which
I. galbana, as a high-quality bait microalgae species, has been widely may be related to the ability of A. macleodii to produce extracellular
used in aquaculture (Tremblay et al., 2007). However, mass cultivation antioxidants (Cao et al., 2021). Whether Marinobacter sp. can produce
of I. galbana is often impaired by different environmental factors. antioxidants is worthy of further study.
When we explored the effects of Marinobacter sp. on the fatty acid
composition of I. galbana, it is interesting to find that the production of
DHA was significantly improved by Marinobacter sp. DHA is one kind of
important nutrient for aquatic animals. It was reported that higher
protein synthesis in microalgae results in elevated expressions of omega-
3/6 fatty acid biosynthetic genes, namely desaturase and elongase,
which subsequently synthesize long-chain PUFAs, such as EPA and DHA
(Jeyakumar et al., 2020). As higher protein synthesis was found in co-
culture, we conjectured that the high protein content might be one
reason for the increased content of DHA. Similarly, the proportion of
unsaturated fatty acids of C. vulgaris-Mesorhizobium sangaii co-culture
was significantly increased compared with the pure algae culture (Wei
et al., 2019). They explained that this enhancement in co-cultures might
be due to the metabolic regulation and nutrient exchange between
microalgae and bacteria.
To further explore the reason for the enhancement of DHA produc­
Fig. 3. Real-time quantitative PCR (RT-qPCR) analysis of the expression of
genes involved in DHA synthesis pathway (ASE2, d4FAD, d5FAD, d6FAD and tion, the effects of Marinobacter sp. on the expressions of genes involved
d8FAD) in co-culture and mono-culture on three time points (2 d, 4 d and 14 d). in DHA synthesis pathway were studied. Our study showed that the
RT-qPCR data represent the mean ± standard error of three independent ex­ expression of ASE2, d4FAD, d5FAD, d6FAD, and d8FAD in the DHA
periments, while asterisks (*) indicate significant difference at p value <0.05. synthesis pathway significantly up-regulated in co-cultures. Therefore,

5
Y.-Y. Wang et al.
the increased content of DHA in co-cultures might be attributed to that
Marinobacter sp. stimulated the expressions of genes involved in the
DHA synthesis pathway. Phosphorus, as a major growth-limiting
nutrient for microalgal growth, is necessary for synthesizing vital bio­
molecules, such as DNA, RNA, and phospholipids (including DHA) (Liu
et al., 2017). Besides, it is interesting to find that Marinobacter sp. has the
ability to decompose and mineralize organic phosphorus. Therefore, we
proposed another hypothesis that Marinobacter sp. decomposed and
mineralized organic phosphorus by the alkaline phosphatase enzyme to
inorganic phosphorus that could be taken up by I. galbana. The increased
phosphorus intake might be the reason for the increased DHA synthesis.
However, additional studies are required to validate this hypothesis.
Marinobacter sp. exists in a wide range of environments, playing an
important in the biochemical cycles of the earth, and it can use a variety
of carbon sources to interact with metals (Singer et al., 2011). It has been
reported that Marinobacter sp. can promote the growth of dinoflagellates
and coccolithophores (Amin et al., 2009b). This Marinobacter strain is
capable of producing vibrioferrin (VF), an unusual lower-affinity dici­
trate siderophore. With the help of VF, the bacteria promote algal
assimilation of iron by facilitating photochemical redox cycling of iron
(Amin et al., 2009a). However, it has been reported that the VF-
producing strains are a small number of strains in the genus Mar­
inobacter, and most strains do not produce VF (Amin et al., 2007; Vraspir
and Butler, 2009). It was worth mentioning that we attempted to
determine the ability to produce VF of this bacterial strain. However, we
did not detect the production of VF in Marinobacter sp. (data not shown).
We also attempted to amplify the VF biosynthetic genes, while we didn’t
obtain the expected PCR product. Therefore, the mechanism for Mar­
inobacter sp. to promote the growth of I. galbana was not dependent on
the VF produced by Marinobacter sp. as reported in dinoflagellates and
coccolithophores. It is well studied that bacteria can secrete hormones,
e.g. indole-3-acetic acid (IAA), that stimulate the growth and meta­
bolism of microalgae (Amin et al., 2015; De Bashan et al., 2008). Our
study showed the potential of Marinobacter sp. to release IAA which
might have a significant positive impact on algal growth (Fig. S4).
Whether Marinobacter sp. enhances the growth of I. galbana by providing
it with IAA merits further study. Taken together, we are sure that the
metabolic regulation and nutrient exchange happened between Mar­
inobacter sp. and I. galbana. What kind of metabolic regulation and what
substances are exchanged merits further investigations.
Collectively, co-cultures of I. galbana and Marinobacter sp. altered the
metabolism and fatty acid contents of microalgal cells and increased the
levels of DHA. Therefore, such an approach could be applied in high-
value compound production and mariculture of microalgae.
5. Conclusion
In our present study, we selected a bacterial strain with a strong
ability to promote microalgal growth and DHA production in I. galbana.
Co-cultures of I. galbana and Marinobacter sp. altered the growth, the
content of chlorophyll a, Fv/Fm, soluble protein content, and SOD ac­
tivity of I. galbana. Besides, the presence of Marinobacter sp. promoted
the production of DHA and up-regulated the expressions of genes
involved in DHA synthesis pathway. Taken together, our current find­
ings provided valuable insights into the interaction between algae and
bacteria and helped improve the yield and quality of microalgae.
CRediT authorship contribution statement
Ying-Ying Wang: Investigation, Data curation, Formal analysis,
Writing – original draft, Writing – review & editing. Si-Min Xu: Data
curation, Writing – review & editing, Validation, Visualization. Jia-Yi
Cao: Funding acquisition, Conceptualization, Supervision, Writing –
original draft, Writing – review & editing. Min-Nan Wu: Data curation,
Writing – review & editing. Jing-Hao Lin: Validation, Visualization.
Cheng-Xu Zhou: Funding acquisition, Conceptualization. Lin Zhang:
6
Aquaculture 556 (2022) 738248
Funding acquisition, Writing – review & editing. Hai-Bo Zhou: Writing
– review & editing. Yan-Rong Li: Writing – review & editing. Ji-Lin Xu:
Funding acquisition, Conceptualization, Supervision. Xiao-Jun Yan:
Conceptualization.
Declaration of Competing Interest
The authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Acknowledgements
This research was supported by the National Key Research and
Development Program of China (2018YFD0900702; 2018YFA0903003;
2018YFD0901504; 2019YFD0900400), Ningbo Science and Technology
Research Projects, China (2019B10006; 2019C10023), Zhejiang Major
Science Project, China (2019C02057), China Agriculture Research Sys­
tem of MOF and MARA, the National Natural Science Foundation of
China (31801724), Natural Science Foundation of Ningbo, China
(2019A610416), the Zhejiang Provincial Natural Science Foundation of
China (LY22C190001) and the Natural Science Foundation of Ningbo
Government (2021 J114).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.aquaculture.2022.738248.
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Another random document with
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members, for given lengths of time, and the surplus funds are
expended in little entertainments such as they like. It is a matter of
much gratification to us in the Burma mission, that the chaplain
selected by the military authorities at present, to fill the post of
Secretary of the Army Temperance Association, is our former
comrade and colleague, the Rev. J. H. Bateson, who in 1887-8 was
with us as Wesleyan Chaplain to the Upper Burma Field Force, and
we heartily wish him success in the work for which he is so well
fitted.
It is a matter of great thankfulness that the Army Temperance
Association is not only fully recognised in our Indian army, but that it
is a standing order that a branch of it has to be maintained in every
regiment and battery. Joining is optional on the part of the men. This
wise course has been amply justified by the results. Sixteen
thousand out of a total of nearly seventy thousand men are enrolled.
It is now found that in proportion as the Army Temperance
Association flourishes, both crime and sickness in the army diminish;
and so far from soldiers needing liquor to sustain them, they are
found far better without it, both in cantonments and in the field. In
fact, it is calculated that every five thousand men in the association
means a battalion of men less in prison and in hospital, and fit for
duty. The wonder is not that such should be found to be the case,
but that it should have taken so many years to find it out. In the
mission we took our stand, of course, on the side of total abstinence,
and embraced every opportunity of advocating this movement, in
military and civil life, both amongst men and amongst women.
We met from time to time with cases of genuine conversion that
gave us great joy. Our Sunday evening English service was always
followed by an after meeting for prayer and exhortation, and made
an opportunity, for any who wished to lead a new life, to give their
hearts to the Saviour. Again and again it was our delight to guide
those who were seeking to do so, at first in Buddhist monasteries,
and pagodas, and anywhere that we could find for the meetings,
often with images of Gautama, and other accessories of Buddhist
worship around, and later on in our own mission school-chapel. I
remember one Sunday evening in particular, the Word came home
to many hearts, and that evening, and in the course of the week, I
had the privilege of close conversation with several, and some of our
Christian members spoke with others who had been awakened by
the influence of the Spirit. Amongst the rest I had a request, through
a soldier, to the effect that Corporal S. would like to talk with me. I
went and met him, and conversed for half an hour in the barrack
yard answering his question, “What must I do to be saved?” The
circumstances of his awakening were peculiar. A certain passage of
Scripture had followed him wherever he had gone. The last Sunday,
just before sailing for India, his mother had requested him to go with
her to the service, and that had been the text. At Malta he had heard
another sermon from the same text. The first time he attended
service after he landed in India it had been the same. And a fourth
time had he heard it preached from at Shwebo in Upper Burma. This
had naturally produced a considerable impression on his mind,
which the sermon of the previous Sunday evening had developed
into decision to serve the Lord. With a little instruction and prayer he
was soon hopefully converted, and happy in the Lord.
It is sometimes urged, as an objection against earnest efforts for
the conversion of sinners, that the results attending such efforts are
not always abiding; but surely no objection could be more illogical or
more ungenerous. If it applies at all, it applies with equal force
against any and every attempt to save men. It may just as well be
alleged against the most formal and perfunctory of ministrations as
against the more direct and strenuous efforts to pluck men out of the
fire. The proper logical outcome of that objection is, “Do nothing at
all.” We might just as well do nothing as make the Gospel a mere
“light to sink by.” A chaplain amongst soldiers must often feel a
painful sense of disappointment at some results of his work, which
are evanescent. The life of the barrack-room necessarily produces,
especially in India, such an artificial condition of things, and involves
such a departure from the Divine ordinance, which is the family, that
it must needs bring with it special trials and stress of temptation to
any of the dwellers there who desire to lead a godly life. Hence every
chaplain has his disappointments over those who grow weary in
well-doing. And yet, on the other hand, such is the principle of
compensation running through the kingdom of grace, that although
barrack-room discipline is bad for the weak Christian, it strengthens
the man of determination, and I question whether there are to be
found anywhere triumphs of saving grace more marvellous than we
find in the army, or more touching examples of humble, sincere and
consistent piety.
We had in the battery of Royal Artillery stationed in Mandalay a
man whose career had been a peculiarly rough one, but who is now
a very bright Christian. He had led a wild life. He was a blacksmith
by trade, and, from his youth up, had been in the habit of spending
all he possibly could in beer, and, as is usually the case, the beer
often made a mere brute and vagabond of him. He first enlisted in a
cavalry regiment, from which, after being often in trouble, he
deserted. For a time he got work, but he still betook himself to the
beer, and the beer made him talk, and let out his former connection
with the army, so that he frequently had to disappear hurriedly, lest
he should be arrested as a deserter. Finding himself in want, he
enlisted again, this time in the Royal Engineers. From this corps he
received his discharge in consequence of an illness. Recovering,
and entering once more on a course of dissipation, he enlisted a
third time, in the Royal Artillery. The Jubilee year gave him the
opportunity to confess his former desertion, and to secure his share
in the general pardon, extended by the Queen to all such cases that
year; and it was not long before the King of kings granted him His
pardon also. Whilst stationed at Woolwich, he happened one
evening, when feeling extremely dejected, to enter the Soldiers’
Home. The Wesleyan chaplain met him there, spoke to him kindly,
and invited him to a meeting. He went. It was a fellowship meeting.
He heard a number of his comrades speak, but so dark was his mind
in reference to religion, that he could not understand them in the
least. However, he gathered that they possessed some source of
comfort and joy within, of which he knew nothing. He followed it up,
became truly converted, and whilst with us in Mandalay lived a most
exemplary life, and exerted a very gracious influence amongst his
comrades. Religion had quickened, as it often does, that once
darkened and besotted nature; and I have seldom met with a better
example of the transforming, elevating power of the Gospel, the
power to keep and sanctify, as well as save.
 Another very satisfactory instance of true conversion, mainly
owing to impressions produced at the parade services, several
Sunday mornings in succession, was that of a pay-sergeant in the
regiment then stationed in Mandalay, a married man living with his
wife and family in the married quarters, a steady, quiet Scotchman,
always well disposed, and of strictly moral life. Parade services are
not always thought to be very good opportunities for getting at the
hearts of soldiers, seeing that they are marched there by
compulsion, not always in the best mood, and with their arms and
accoutrements (in India), which is a different thing from going to a
voluntary service off parade. But does not this fact challenge, as it
were, the chaplain to give them of his brightest and best? It must,
before all, be very short, or he will ruin everything, and send them
away worse than they came; something short, lively and heart-
stirring, full of Christ, full of apt illustration, and full of sympathy with
souls, so that he may capture these soldier lads in spite of
themselves. Well, it was at these parade services that Sergeant C.
felt his mind awakened to new views of truth and duty and Christian
privilege. Being aroused about the matter he attended also the
evening services, and the devotional meetings on the week-nights,
and soon got the light he required, and found himself a new man in
Christ Jesus. Well conducted and steady as he had been before, his
conversion nevertheless made a great difference to him, giving
clearness and brightness to his religious character, and kindling in
him a new zeal for the conversion of others.
We found drink to be a fearful curse, not only amongst the English
residents, but amongst the natives also. I had a servant, a native of
India. He was a great gambler, and very lazy, dishonest and
troublesome altogether. Bad as he was, we bore with him over two
years, fearing that if we discharged him we might have to put up with
somebody worse. Just before we left Burma we dismissed him,
because he had sent away his wife and taken up with another. Since
coming to England, I learn that this man, in a fit of drunkenness,
murdered this woman, with circumstances of unusual atrocity, and
that he had to suffer the extreme penalty of the law.
 But this drink monster is no respecter of persons, and makes no
distinction of race, sweeping down all before it without any
discrimination. An English soldier in Mandalay, who had been an
abstainer for a considerable period, suddenly took to liquor again
one day, and got drunk. That evening he took out his rifle, put in a
cartridge, walked down out of the bungalow, and took the direction
that the seven devils within him pointed out. This happened to be
towards the sergeants’ mess, a separate building a stone’s-throw
away. That evening, a party of sergeants were enjoying a festive
gathering, in honour of the seventeenth anniversary of the enlistment
of one of their number. His health had just been proposed, and he
stood up to reply, when at that very moment the poor crazed
drunkard outside fired, and shot the sergeant dead. There had been
no provocation, and no reason could be assigned for the rash act. It
was merely “the drink.” In the distant future, when the temperance
reform shall have won its way, and the customs of English society
shall have undergone a great change, people will greatly wonder that
their forefathers took so long to discover that liquor was their enemy,
and not their friend.
Another example, and I bring these reminiscences to a close. It is
the case of a soldier, who formerly belonged to a cavalry regiment,
stationed at the time I speak of at one of the principal military
stations in the south of India. He had plunged into drinking and vice,
until at last he was told by the doctor that he had gone as far as his
constitution would allow him, and that if he went any further it would
be the end of him. This weighed upon his mind, and a deep sense of
his sinfulness and a desire for better things resulted. He felt he
needed Divine help, and he thought he had better begin again to
pray, a thing he had long ceased to do. But how to begin in a
barrack-room, where many pairs of eyes would see him, and
misunderstand, and ridicule him? Well, he would wait until all was
quiet, and then kneel down by his cot and pray. He waited, but as he
was musing the fire kindled, and when he did begin to pray, so
urgent was his pleading with God for mercy, that his voice rang
through the barrack-room, and all his comrades were aroused by it.
They thought he was mad. He was removed to the guard-room, and
put under restraint. There, in the quietude of that solitary place, he
found pardon, and his soul was filled with peace. Next day the
medical officer saw him; he could not quite make out the case, but
adopted the safe course of keeping him still under restraint. He told
the doctor what it was that had caused the trouble of his mind, and
how he had gained deliverance, adding that if they had said he was
mad before, it would have been quite true, but that now he had come
to his right mind. This explanation only induced the man of science
dubiously to elevate his eyebrows. It was a kind of case he was not
familiar with. Though perfectly sane, and calm and happy, he was
kept under restraint for a month, and he was accustomed to say that
that month, almost entirely alone with God and his Bible, was the
happiest period of his life.
That work of grace, so strangely begun, was thorough and
abiding. It was well known in the station, and produced a great
impression for good, supported as it was by his subsequent
consistent conduct. It was years after his conversion that I knew him
intimately in Burma as a non-commissioned officer, serving in an
important and responsible military position, for which he had been
specially selected; and I knew him for several years as a consistent
Christian, whose firm example—and happy, cheerful character made
him a blessing to others, and who was never backward in quietly and
judiciously speaking for the Master.
 CHAPTER XIX.
A JUNGLE JOURNEY.

We had a great desire in the mission to pay a visit to the Chin

tribes on the western frontier, with the view of ascertaining their
locality and circumstances. Consequently, when the cool season
arrived, the usual time of year for tours, I started with my brother
missionary, Mr. Bestall, then stationed at Pakokku, for a journey to
the Chin country. The Chins, as explained in Chapter X., are not
Buddhists, but worshippers of spirits or demons, and are on that
account more barbarous, and, contradictory as it may seem to say
so, more ready of access than the Buddhistic races, to Christian
mission effort.
We left Pakokku at 4 a.m. one morning in November, mounted on
two lively Burmese ponies, with a cart drawn by a pair of bullocks for
our things. In travelling through the jungle you need to take almost
everything you require; so that when the sugar, and salt, and tea,
and bread, and butter, and tins of meat (in case the gun brings down
nothing), and soap, and candles, and rice, and curry, and frying-pan,
and kettle, and crockery, and a few other simple necessaries, not
omitting medicines, are packed up in a box, and the pillows and rugs
for the night rolled up in a mat, and a change or two of clothes put in
a portmanteau, and a few Gospel portions and tracts included, to
distribute in the villages as we pass along, you find you require a cart
to carry them. As the cart can only travel, on an average, about two
miles an hour, it requires a long day to do twenty miles. There is no
advantage in going ahead of the cart, you only have to wait at the
end of the stage, and it is more enjoyable to spend the time in
leisurely travelling. In this way these jungle journeys, though the
travelling is rough and often fatiguing, are very serviceable for the
sake of the change of air and scene, and the free out-door exercise
they afford.
 We managed to make a stage of six miles before it was daylight,
and pushed on the next stage of fifteen miles farther without
stopping. There we halted at one of the establishments constructed
at every stage along this route, to accommodate troops and
convoys, on the march to the military and police stations on the
frontier. It consisted of long rows of temporary bamboo barracks, and
a bamboo shed for the officers. There was a Burmese police guard
close by. How regularly and irreproachably that Burman constable
shouldered his rifle, and did his “sentry go,” while we were looking
on! Here we halted for our midday meal and a short rest. It is
wonderful with what dexterity your native servant, availing himself of
almost no facilities for cooking, can produce you a savoury breakfast
on the march. Three stones or bricks to support the kettle, and the
same for the frying-pan, are all he requires for a fireplace, and a few
sticks and bits of bamboo out of the jungle are enough for a fire. In
the afternoon we did another twelve miles, making thirty-three miles
that day, which was rather more than the cartman would have driven
his bullocks, if it had not been that the next day was Sunday, the day
of rest. The weather was lovely, being the best time of the year for a
journey. It was not much hotter in the day than a warm summer day
in England; the nights and mornings were chilly.
At Pyinchaung we halted on the Saturday night, again putting up in
the temporary military lines. The route we had taken was not a road,
as we understand roads in England, but, strictly speaking, more of a
track, fairly passable in dry weather for carts, but almost
impracticable after heavy rains. Good metalled roads are a luxury we
have not seen much of as yet in Upper Burma, but we shall get them
in course of time. On the Sunday we rested, and spent some time
amongst the villagers, distributing tracts and preaching. Here we had
the misfortune to lose our two ponies. Mr. Bestall, pitying them, that
there was so little grass to eat in the rest-house enclosure, opened
his kind heart, and the gate at the same time, and let them out to
graze, giving them in charge to the cartman to look after. He followed
them for awhile, and then, native-like, came back without them. We
went in search of them, but never saw them again that journey. They
strayed for many miles, and it was a month before they were brought
back. It speaks well for the hold the English now have on the
country, and the great diminution of crime, that search was made in
all the district round, and they were returned by the police, as we felt
sure they would be. We had to borrow for the rest of the way.
An eloquent reminder of the troublous times we had then barely
passed through, was the little police fort close by the rest-house,
constructed on the top of a ruined pagoda, where there was a view
of the country for some distance round. During the first three or four
years of British rule places like this had to be selected wherever
practicable, and made strong enough to stand a rush by dacoits, and
a careful watch had to be kept. The little bamboo house was perched
right on the top of the steep mound of ruined brickwork, a wall breast
high was erected round it, and with a few resolute men, well armed,
inside, it would not have been easy to take. The rest-house at
Pyinchaung, looking westward, overlooked a most lovely valley of
great extent and fertility, through which we had now to pass. This
valley looks as if it might have been at one time the bed of a mighty
river, but the stream is now contracted to a very narrow span, and
the alluvial soil of this rich valley is a veritable land of plenty. I never
saw a region more lovely with “the fairer forms that cultivation glories
in” than the Yaw valley then appeared. Whilst crossing this great
valley we rode through fields of maize, and another tall grain, a kind
of millet, far above our heads, with rice fields here and there, and
abundance of pumpkins, beans and other vegetables, the ponies
snatching an occasional bite at the sweet juicy stems and leaves of
the millet, which they are so fond of. Carts in great numbers passed
us, drawn by well-fed, plump oxen, and mostly laden with the leaves
which envelop the maize cobs, all laid straight and packed neatly in
bundles. The maize crop had nearly all been reaped, and these
leaves of the Yaw valley maize, not the grain itself, form the most
valuable product of the crop, and are largely sold all over Burma for
the purpose of enveloping Burmese cigars.
The people of this district all looked fat and well fed. There was
abundance of cattle, and the inhabitants of that region seemed to
want for nothing in a material point of view. Fifteen miles from
Pyinchaung we reached Pauk, a small Burmese town, the
headquarters of the township officer, a police officer, and a lieutenant
in charge of a detachment of Madras troops. They were all very
young Englishmen, two of them apparently not over twenty-five, and
it might have seemed odd at first sight to see such young men in
such responsible positions. But suddenly having to find a sufficient
staff of officials, to rule over a country as large as France, has
involved engaging the services of many young men, for they must
enter upon their duties young to be properly trained for the work; and
it is a notable fact that the great work of pacifying and restoring to
order Upper Burma has been chiefly the work of very young men.
The civil officer is a magistrate, and has to try such cases as are
within his jurisdiction, to collect the revenue through his native
subordinates, to keep his eye on everything in general, and to keep
the Deputy Commissioner of the district informed of all that is going
on, to initiate whatever is needful for the well-being of the
community, and to act the part of a father to the people of his
township, which is as large, though not so populous, as an English
county. The police inspector is responsible for the maintenance of
order, and the pursuit and arrest of criminals; and the military may at
any time be called out to take the field, and try conclusions with
some dacoit band that has gathered in force. As far as I could judge
they all seemed very fit for the work they had to do.
Only a year or so before, this township of Pauk was in an
exceedingly disturbed state, by reason of dacoit bands; and if things
had not greatly improved, we could never have travelled unprotected
through it as we did. It is to the credit of these young men, and the
troops and police under them, that things are so peaceable now. We
saw at Pauk the same abundant evidences of prosperity and
improvement, that are visible everywhere throughout the country, not
only in the erection of a new court house and public Government
office, and many private houses, but still more in the great
improvements made about the town, in the improvement of the
roads, and most of all in the construction of a new bazaar, which the
township officer showed us through with pardonable pride, and in
which a great deal of business was going on.
Having stayed in Pauk the night, we were off the next morning
early, forded the river, travelled a stage, and rested for our midday
meal in a monastery. This is no uncommon thing in Burma; we had
occasion to do so several times on this journey. There is hardly a
village without its monastery, one or more, always the best building
in the place, and kept very clean; and it generally happens, as in this
case, that there are some vacant buildings used as rest-houses by
chance travellers. We always found the monks affable and pleasant
to meet, quite chatty, with a kind of friendly familiarity entering at
once into conversation, and evidently not having the slightest
objection to seeing us about the premises. We, on our part,
reciprocated these advances, and made things pleasant all round.
Thus we travelled on from day to day, as fast as our bullocks could
make the journey, which was very slowly indeed. As we could gain
nothing by going ahead of our cart, we were obliged to spend the
spare time as best we could. My companion, having a gun, and
being a good shot, managed to get something every day, which, in
the entire absence of the butcher’s shop, was very acceptable for
the larder. We had either a hare, wild pigeons, jungle fowl,
partridges, or something. Game is abundant on the route, and in the
jungle you have no fear of encroaching on anybody’s preserves.
I suppose there are few places in the world now, however remote,
where an Englishman is not constantly meeting with some
wandering specimens of his countrymen; and even in the wildest
recesses of the jungles of Upper Burma, you are liable to the
discovery that the genus Englishman includes the species
gentleman and the species snob. We had a curious illustration of
this. Arriving one evening, long after dark, about eight o’clock, at a
roadside rest-house, built by the Public Works Department for the
common use of English travellers, we found a gentleman whom I will
call Captain X. He was travelling in charge of a large military convoy
of elephants, ponies and other baggage animals, carrying up
supplies to one of the distant military stations in the Chin country. He
and a junior companion were in possession of the comfortable,
spacious, three-roomed bamboo rest-house, where there would
have been ample accommodation for us as well, with our scanty
travelling kit, in the third room, which they were not using; and they
had really no more right to monopolise the whole than we had.
However, they were in possession; and on our presenting ourselves
at the door, Captain X. never so much as asked us to step inside,
never attempted even to ascertain who we were, or to enter at all
into conversation with us, but simply directed our attention to a dirty,
shabby bamboo shed at the lower part of the compound, built for
natives, and at that moment quite full of Burman coolies, who, he
cheerfully assured us, would readily “nip out” and make room for us,
if we asked them. Some people’s idea of the purpose of other people
seems to be that they were meant by Providence to “nip out” and
make way for them! Gathering from his manner that he did not mean
us to have the use of the vacant room at the rest-house, or to show
the slightest courtesy in any way, we betook ourselves to the said
outbuilding. The courteous Burmans squeezed themselves into
smaller space, and left us enough room to spread our rugs on the
bamboo floor, and we managed to put up for the night. I am glad to
say that this kind of discourtesy is very uncommon indeed abroad. In
all my experience of many years in Ceylon and Burma, I have never
met with such scant civility from a fellow-traveller in the jungle, but
always something more in accordance with the circumstances.
A striking contrast to this was the gentlemanly conduct of
Lieutenant T., whom we happened to meet in a similar way at a rest-
house on the return journey. He was in possession, too, before we
arrived, and though that rest-house only consisted of one room,
which he was occupying, he most kindly pressed us to share it with
him. This, however, we would not do, but decided to occupy a zayat
which we found vacant close by. We entered into conversation, and
shortly after, when we took leave of him for the night, we found that,
finding our cart had not arrived, and that our supper would have
been long delayed, he had sent his servant boy round with enough
supper for both of us, and a candle by the light of which to eat it!
Lieutenant T. is a brave man, and has made quite a name in
connection with the rough military and civil work of the last few
years, amongst the tribes of the western frontier. He has been, as we
were elsewhere informed, in thirteen engagements; and he was at
that time returning to his distant appointment on the hills, from an
event which must have been to him, as a soldier, the proudest
moment of his life, when the general decorated him, in the presence
of all the troops of the station assembled on parade, with the
Distinguished Service Order. It was currently reported by his brother-
officers that it would have been the Victoria Cross, had he not been
in command of the detachment on the special occasion, and as the
writer of the despatch, he chivalrously gave the praise to another.
I could not but observe this, as another instance, showing that true
bravery is usually associated with true modesty, and all other
gentlemanly qualities.
We now began to make our way over a mountainous ridge, along
which an earthen road had been cut out, but not gravelled, and many
a rustic bridge erected over the torrents that crossed the track, by
the British, about a year before, on purpose for the Chin Expedition,
and for subsequent traffic. Some £35,000 we were told had been
spent upon it. Some thousands of coolies were brought over from
India, and the thing was done without delay. The expedition would
have been almost impossible without a road. This serves to give the
reader a glimpse of what it means to undertake the pacification and
administration of a new country of abundant resources, but without
means of communication, and with much raiding and dacoity going
on. A road, or still better a railway, always means increased traffic
and commerce, better markets for produce, and better means of
getting about, and is itself, therefore, a pacifier and civiliser of no
mean account; and it soon tends, under British law and insistence on
good behaviour, to demonstrate that honesty is the best policy. Good
government should always make it pay better to lead an honest,
industrious, orderly life, than to pursue a career of robbery and
violence—should, in fact, make it hard to do wrong and easy to do
right.
This part of our journey was through a hilly and picturesque
country, consisting almost entirely of thick natural forest, with many
teak and other fine timber trees, and bamboo jungle everywhere. At
Thileng we were ninety-eight miles from Pakokku, and close to the
Chin Hills. We had here to leave the road, and our cart could go no
farther with us, as the hills are very precipitous, and there is only a
jungle path. We therefore reduced our baggage to the lowest
possible limits of sheer necessity, and had our few things carried the
remainder of the journey by a couple of coolies.
We observed that the village of Thileng had some attempts at
protection against the Chin raids. At each of the four ends of the
village, where the two main roads, placed at right angles, lead out to
the jungle, there are log huts erected, where a police guard can be
sheltered against their arrows and spears, and the gates are shut at
night. The remaining protection consists of a broad hedge of dead
thorns heaped all round the village. At this and several villages in the
vicinity sad tales were told us of Chin raids, in which Burmans were
taken captive, and some of them detained amongst the Chins for
many years. The various British expeditions sent up to the Chin
tribes, with a view of reducing them to order, have released from
time to time a great many of these unfortunate people, and the
practice will soon come to an end, if it has not already ceased. This
is one example of the ways in which English rule is a great blessing
to a country like Burma, in removing such an intolerable burden as
this constant dread of these murderous and disastrous raids, and the
subsequent miseries of the unfortunate captives. The distance from
Thileng to Pinloak, the nearest Chin village, is about sixteen miles,
and over as rugged and difficult a path as ever I travelled. About
noon we halted, and had our lunch at the bottom of a very lovely
gorge, by the side of an icy-cold stream, just the picturesque kind of
place that would become a favourite with tourists in England.
At about two o’clock we approached the village, and we halted,
under cover of the tall grass, while our Burman guide went forward to
announce our approach. Presently they called to us to come forward,
and we emerged from the tall grass upon a clearing, on the steep
hillside, of several acres. The forest trees and undergrowth had been
felled and burnt, and crops of various kinds of grain, cultivated in a
rough and ready manner, and a few vegetables, were growing. The
people received us in a friendly way, and we went forward and
rested in the nearest house, which was of bamboo, something like
the houses of the Burmans. We found the Chins in many respects
different from the Burmans—far more backward in civilisation. In
colour they are about the same complexion, a light brown, but
altogether dirty and unwashed. The men wear the merest rag of a
garment, the women wear a kind of tunic covering the body, but the
legs and thighs and feet are quite bare. The peculiar custom of
tattooing the faces of the women, described in Chapter X., gives
them rather a hideous appearance, and when seen in such a dress,
with the face tattooed in that fashion, and with a bamboo pipe stuck
in their mouths, smoking, the effect is not the most lady-like
imaginable. Still it is only fair to say that the women we saw, despite
all these disadvantages, did not strike us as looking particularly
unwomanly. Some of the faces, both of the men and women, were of
rather a fine cast, notwithstanding their barbarous, unkempt
appearance; but the greater part of them wore that degraded
appearance which utter ignorance and the many hardships of a
savage life generally produce. As I have given many particulars
about the manners and customs of the Chins in a former chapter, I
need not repeat it. We spent the afternoon fraternising with them in
their houses, making purchases of some of their weapons and other
articles, which they certainly did not make the mistake of charging
too little for, and witnessing their wonderfully accurate shooting with
the bow and arrow.
 “THE WOMEN AND GIRLS OF THE CHIN TRIBE WEAR A KIND OF TUNIC.”

As evening drew on our Burmese guide advised us to camp out

across the river in preference to sleeping in the village. As the best
native houses in Burma are apt to harbour much vermin, and as the
Chins never think of such a thing as washing their bodies, it may be
understood that we were not unwilling to take that advice. Moreover,
it was desirable to avoid any complications that might lead to a
breach of the peace, for with barbarians it is sometimes a word and
a blow, and the blow first. We therefore crossed the river and
prepared to camp out in the forest, under a great clump of bamboos,
spreading our mats on the sand, and kindling a good fire, for it
became very cold as the night advanced, and the dew dropped from
the trees almost like rain. Some of the Chins came over and sat with
us as we ate our supper, accepting a taste of each article, and
testifying their approval, especially of the jam. As one or two of them
could talk Burmese we were able to converse with them, and until
late at night they stayed listening round our camp fire, as we told
them about England and its greatness, and tried to explain, as well
as we could make them understand, some of the leading truths of
Christianity.
I must not omit to state what it was in us that astonished them
most of all. After they had investigated the mystery of the gun, and
had fired off a cartridge, and had examined whatever else we had
about us that was curious, my companion suggested to me that, as I
embodied in my own person a good example of the dentist’s art, it
might be well to let them see what the English experts could do in
supplementing deficiencies of that nature. I thought it was a good
suggestion; so calling their special attention to what I was about to
do, I quietly detached the upper set of teeth and held it forth at arm’s
length, full in the gaze of the astonished barbarians, and then slipped
it back again in a moment, and showed them that I was able to eat
with them just as well as they could with theirs. We had expected
them to be surprised at this exhibition, but their astonishment
exceeded our expectation. Up to that moment my friend, as the
proprietor of the gun, and the more affable and engaging gentleman
of the two, had been the chief centre of observation and admiration,
but after that he had to take the second place. They were greatly
amazed. Never had they seen such a thing before. They had no idea
it was possible to do it. To make a gun, or any other piece of
mechanism, or any manufactured article, was very likely within the
power of a highly civilised people. But to be able to detach and take
out the whole upper set of teeth, gums, palate and all (apparently),
and then to slip them in again, and enjoy the full and perfect use of
them!—that far exceeded any notions they had previously formed of
what was possible, and they evidently regarded this not as a piece of
mechanism, but more in the light of an utterly inexplicable, if not
magical, accomplishment.
It is not amiss for barbarous people like these, who have been
accustomed to set all law and order at defiance, and raided upon
Burmese territory just as they liked, to have the opportunity of seeing
for themselves some marks of a superior civilisation. It may be
expected to induce in them a wholesome dread of the British power,
and a more orderly and peaceful mode of life.
They begged us to stay over the next day, stating that they wished
to bring their people from far and near to see this strange sight, but
the risk of fever, through sleeping out in the jungle, was too great to
justify us in remaining longer, and next day we left Pinloak, and
returned the way we came. And we live in hopes that, when the
funds will allow of it, the information we gleaned on this tour may be
turned to good account.